Ligand for the c-kit receptor and methods of use thereof

ABSTRACT

A pharmaceutical composition which comprises the c-kit ligand (KL) purified by applicants or produced by applicants&#39; recombinant methods in combination with other hematopoietic factors and a pharmaceutically acceptable carrier is provided as well as methods of treating patients which comprise administering to the patient the pharmaceutical composition of this invention. This invention provides combination therapies using c-kit ligand (KL) and a purified c-kit ligand (KL) polypeptide, or a soluble fragment thereof and other hematopoietic factors. It also provides methods and compositions for ex-vivo use of KL alone or in combination therapy. A mutated KL antagonist is also described. Such an antagonist may also be a small molecule. Antisense nucleic acids to KL as therapeutics are also described. Lastly, compositions and methods are described that take advantage of the role of KL in germ cells, mast cells and melanocytes.

[0001] This invention is a continuation-in-part application ofPCT/US91/06130, filed Aug. 27, 1991, which is a continuation-in-part ofU.S. Ser. No. 549,306, filed Oct. 5, 1990, which in turn is acontinuation-in-part of U.S. Ser. No. 573,483, filed Aug. 27, 1990, nowabandoned, the contents of all three are hereby incorporated byreference into the present application.

[0002] The invention described herein was made in the course of workunder Grant No. RO1-CA 32926 and ACS MV246D from the National Instituteof Health and American Cancer Society, respectively. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Throughout this application various publications are referred byarabic numerals to within parenthesis. Full bibliographic citations forthese references may be found at the end of the specificationimmediately preceding the claims. The disclosures for these publicationsin their entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thisinvention pertains.

[0004] The c-kit proto-oncogene encodes a transmembrane tyrosine kinasereceptor for an unidentified ligand and is a member of the colonystimulating factor-1 (CSF-1)—platelet-derived growth factor (PDGF)—kitreceptor subfamily (7, 41, 57, 23). c-kit was recently shown to beallelic with the white-spotting (W) locus of the mouse (9, 17, 35).Mutations at the W locus affect proliferation and/or migration anddifferentiation of germ cells, pigment cells and distinct cellpopulations of the hematopoietic system during development and in adultlife (47, 51). The effects on hematopoiesis are on the erythroid andmast cell lineages as well as on stem cells, resulting in a macrocyticanemia which is lethal for homozygotes of the most severe W alleles(46), and a complete absence of connective tissue and mucosal mast cells(72). W mutations exert their effects in a cell autonomous manner (28,46), and in agreement with this property, c-kit RNA transcripts wereshown to be expressed in targets of W mutations (35). High levels ofc-kit RNA transcripts were found in primary bone marrow derived mastcells and mast cell lines. Somewhat lower levels were found inmelanocytes and erythroid cell lines.

[0005] The identification of the ligand for c-kit is of greatsignificance and interest because of the pleiotropic effects it mighthave on the different cell types which express c-kit and which areaffected by W mutations in vivo. Important insight about cell typeswhich may produce the c-kit ligand can be derived from the knowledge ofthe function of c-kit/W. The lack of mast cells both in the connectivetissue and the gastrointestinal mucosa of W/W^(v) mice indicated afunction for c-kit in mast cell development. Mast cells derived frombone marrow (BMMC) are dependent on interleukin 3 (IL-3) and resemblemast cells found in the gastrointestinal mucosa (MMC) (92, 93).Connective tissue mast cells derived from the peritoneal cavity (CTMC)in vitro require both IL-3 and IL-4 for proliferation (79, 75). Theinterleukins IL-3 and IL-4 are well characterized hematopoietic growthfactors which are produced by activated T-cells and by activated mastcells (92, 94, 95, 96, 97). An additional mast cell growth factor hasbeen predicted which is produced by fibroblasts (47). In the absence ofIL-3, BMMC and CTMC derived from the peritoneal cavity can be maintainedby co-culture with 3T3 fibroblasts (98). However, BMMC from W/W^(v) miceas well as mice homozygous for a number of other W alleles are unable toproliferate in the fibroblast co-culture system in the absence of IL-3(99, 100, 38). This suggested a function for the c-kit receptor inmature mast cells and implied that the ligand of the c-kit receptor isproduced by fibroblasts. Huff and coworkers recently reported thestimulation of mast cell colonies from lymph node cells of mice infectedwith the nematode Nippostronglyus brasiliensis by using concentratedconditioned medium from NIH 3T3 fibroblasts (84). A short term mast cellproliferation assay was developed which means to purify a fibroblastderived activity (designated KL) which, in the absence of IL-3, supportsthe proliferation of normal BMMC's and peritoneal mast cells, but notW/W^(v) BMMC's. In addition, KL was shown to facilitate the formation oferythroid bursts (BFU-E). The biological properties of KL are inagreement with those expected of the c-kit ligand with regard to mastcell biology and aspects of erythropoiesis. The defect W mutations exertis cell autonomous; in agreement with this property, there is evidencefor c-kit RNA expression in cellular targets of W mutations (35, 39).The recent characterization of the molecular lesions of several mutantalleles indicated that they are loss-of-function mutations that disruptthe normal activity or expression of the c-kit receptor (35, 100, 101,36).

[0006] Mutations at the steel locus (S1) on chromosome 10 of the mouseresult in phenotypic characteristics that are very similar to those seenin mice carrying W mutations, i.e., they affect hematopoiesis,gametogenesis, and melanogenesis (5, 47, 51). Many alleles are known atthe S1 locus; they are semidominant mutations, and the different allelesvary in their effects on the different cell lineages and their degree ofseverity (47, 51). The original S1 allele is a severe mutation. SIISIhomozygotes are deficient in germ cells, are devoid of coat pigment, anddie perinatally of macrocytic anemia (5, 50). Mice homozygous for the S1allele, although viable, have severe macrocytic anemia, lack coatpigment, and are sterile. Both SII⁺ and S1^(d)/+heterozygotes have adiluted coat color and a moderate macrocytic anemia but are fertile,although their gonads are reduced in size. In contrast to W mutations,S1 mutations are not cell autonomous and are thought to be caused by adefect in the micro-environment of the targets of these mutations (28,30, 12). Because of the parallel and complementary characteristics ofmice carrying S1 and W mutations, we and others had previouslyhypothesized that the S1 gene product is the ligand of the c-kitreceptor (51, 9).

[0007] The proto-oncogene c-kit is the normal cellular counterpart ofthe oncogene v-kit of the HZ4—feline sarcoma virus (7). c-kit encodes atransmembrane tyrosine kinase receptor which LS a member of the plateletderived growth factor receptor subfamily and is the gene product of themurine white spotting locus (9, 17, 23, 35, 41, 57). The demonstrationof identity of c-kit with the W locus implies a function for the c-kitreceptor system in various aspects of melanogenesis, gametogenesis andhematopoiesis during embryogenesis and in the adult animal (47,51). Inagreement with these predicted functions c-kit mRNA is expressed incellular targets of W mutations (3, 24, 25, 35, 39).

[0008] The ligand of the c-kit receptor, KL, has recently beenidentified and characterized, based on the known function of c-kit/W inmast cells (2, 14, 37, 38, 56, 58, 59). In agreement with theanticipated functions of the c-kit receptor in hematopoiesis KLstimulates the proliferation of bone marrow derived and connectivetissue mast cells and in erythropoiesis, in combination witherythropoietin, KL promotes the formation of erythroid bursts (day 7-14BFU-E). Furthermore, recent in vitro experiments with KL havedemonstrated enhancement of the proliferation and differentiation oferythroid, myeloid and lymphoid progenitors when used in combinationwith erythropoietin, GM-CSF, G-CSF and IL-7 respectively suggesting thatthere is a role for the c-kit receptor system in progenitors of severalhematopoietic cell lineages (27, 37).

[0009] Mutations at the steel locus on chromosome 10 of the mouse resultin phenotypic characteristics that are very similar to those seen inmice carrying W mutations, i.e., they affect hematopoiesis,gametogenesis and melanogenesis (5, 47, 51). The ligand of the c-kitreceptor, KL, was recently shown to be allelic with the murine steellocus based on the observation that KL sequences were found to bedeleted in several severe S1 alleles (11, 38, 59). In agreement with theligand receptor relationship between KL and c-kit, S1 mutations affectthe same cellular targets as W mutations, however, in contrast to Wmutations, S1 mutations are not cell autonomous and they affect themicroenvironment of the c-kit receptor (12, 28, 30). Mutations at thesteel locus are semidominant mutations and the different alleles vary intheir effects on the different cell lineages and their degree ofseverity (47, 51). The original S1 allele is an example of a severe S1mutation. S1/S1 homozygotes are deficient in germ cells, are devoid ofcoat pigment and they die perinatally of macrocytic anemia (5,50). Micehomozygous for the S1^(d) allele, although viable, have severemacrocytic anemia, lack coat pigment and are sterile (6). Both S1/+ andS1^(d)/+ heterozygotes have a diluted coat color and a moderatemacrocytic anemia, but they are fertile, although their gonads arereduced in size. Southern blot analysis of S1d/+ DNA by using a KL cDNAas a probe indicated an EcoR1 polymorphism, suggesting that thismutation results from a deletion, point mutation or DNA rearrangement ofthe KL gene (11).

SUMMARY OF THE INVENTION

[0010] A pharmaceutical composition which comprises the c-kit ligand(KL) purified by applicants or produced by applicants' recombinantmethods in combination with other hematopoietic factors and apharmaceutically acceptable carrier is provided as well as methods oftreating patients which comprise administering to the patient thepharmaceutical composition of this invention. This invention providescombination therapies using c-kit ligand (KL) and a purified c-kitligand (KL) polypeptide, or a soluble fragment thereof and otherhematopoietic factors. It also provides methods and compositions forex-vivo use of KL alone or in combination therapy. A mutated KLantagonist is also described. Such an antagonist may also be a smallmolecule. Antisense nucleic acids to KL as therapeutics are alsodescribed. Lastly, compositions and methods are described that takeadvantage of the role of KL in germ cells, mast cells and melanocytes.

[0011] This invention provides a nucleic acid molecule which encodes anamino acid sequence corresponding to a c-kit ligand (KL) and a purifiedc-kit ligand (KL) polypeptide.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1. Proliferative response of +/+ and W/W^(v) BMMC tofibroblast conditioned medium and IL-3. Mast cells derived from +/+ orW/W^(v) bone marrow were cultured in the presence of 1% 3 CM, 10% FCM(20× concentrated), or medium alone. Incorporation of ³H-thymidine wasdetermined from 24-30 hours of culture.

[0013]FIG. 2. Chromatographic profiles of the purification of KL.

[0014] A. Gel filtration chromatography on ACA 54 Ultrogel. Absorbanceat 280 nm is shown by a broken line and bio-activity by a solid line.The position of the elution of protein size markers is indicated in kD.

[0015] B. Anion exchange FPLC on a DEAE-5PW column. The NaCl gradient isindicated by a dotted line.

[0016] C. Separation on semi-preparative C18 column. The 1-propanolgradient is indicated by a dotted line.

[0017] D. Separation on analytical C18 column.

[0018]FIG. 3. Electrophoretic analysis of KL. Material from individualfractions was separated by SDS/PAGE (12%) and stained with silver. Theposition of KL (28-30 kD) is indicated by an arrow. KL activity ofcorresponding fractions is shown below.

[0019] A. Analysis of 0.5 ml fractions from analytical C18 column elutedwith ammonium acetate buffer and 1-propanol gradient.

[0020] B. Analysis of 0.5 ml fractions from analytical C4 column elutedwith aqueous 0.1% TFA and absence of 2-mercapto-ethanol.

[0021]FIG. 4. Proliferation of W^(v) mutant mast cells in response toKL. Mast cells were derived from individual fetal livers fromW/+×W/+mating, or bone marrow of wildtype, W^(v) and W⁴¹ heterozygotesand homozygoses. The proliferation characteristics of mutant mast cellswas determined by using increasing concentrations of KL in aproliferation assay. Homozygous mutant mast cells are indicated by asolid line, heterozygotes mutant mast cells by a broken line andwildtype mast cells by a dotted line, except for W where normal fetusesmay be either +/+or W/+.

[0022]FIG. 5. Comparison of c-kit expression and growth factorresponsiveness in BMMC and peritoneal mast cells (CTMC/PMC).

[0023] A. Fluorescent staining of heparin proteoglycans in purified PMCand BMMC by using berberine sulfate.

[0024] B. Determination of c-kit cell surface expression in PMC and BMMCby FACS using c-kit antibodies. Anti-c-kit serum is indicated by a solidline and non-immune control serum by a dotted line.

[0025] C. Determination of the proliferation potential of PMC to KL.5000 cells were plated in 0.5 ml, in the presence of 1000 U/ml of KL,10% Wehi-3CM or RPMI-C alone and the number of viable cells wasdetermined two weeks later.

[0026]FIG. 6. Determination of burst promoting activity of KL. Bonemarrow and spleen cells were plated in the presence of erythropoietin(2U/ml) and pure KL was added at the concentrations shown. The number ofBFU-E was determined on day 7 of culture. This data represents the meanof two separate experiments, each with two replicates per concentrationof KL.

[0027]FIG. 7. Determination of KL dependent BFU-E formation from W/Wfetal livers. Fetuses from mating W/+ animals were collected at day 16.5of gestation. One fetus out of four was a W/W homozygote. Liver cellswere plated at 10⁵ cells/ml in the presence of either control medium,IL-3 (50 U/ml) or KL (2.5 ng/ml). All cultures contained erythropoietin(2U/ml). Data is expressed as the number of BFU-E/liver and is the meanof 2 replicate plates. The data for +/+ or W/+ fetuses is the mean fromthe three normal fetuses in the liver.

[0028]FIG. 8. N-terminal amino acid sequence of KL and deduction of thecorresponding nucleic acid sequence by PCR. Top line: N-terminal aminoacid sequence (residues 10-36) of KL. Middle Line: Nucleotide sequencesof three cDNAs obtained by cloning the 101 bp PCR product (see FIG. 10)into M13 and subsequent sequence determination. Bottom Line: sequencesof the degenerate sense and antisense primers used for first-strand cDNAsynthesis and PCR. The amino acid sequence also is identified as SEQID:NO:2.

[0029]FIG. 9. Northern blot analysis using the PCR generatedoligonucleotide probes corresponding to the isolated c-kit ligandpolypeptide. A 6.5 kb mRNA was isolated with labelled probes.

[0030]FIG. 10. Derivation of cDNAs corresponding to the N-terminal aminoacids 10-36 of KL by RT-PCR. One microgram of poly(A)⁺ RNA from BALB/c3% 3 cells was used as template for cDNA synthesis and subsequent PCRamplification in combination with the two degenerate oligonucleotideprimers. Electrophoretic analysis of the 101 bp PCR product in agaroseis shown.

[0031]FIG. 11. Nucleotide Sequence and Predicted Amino Acid Sequence ofthe 1.4 kb KL cDNA clone. The predicted amino acid sequence of the longopen reading frame is shown above and the nucleotide sequence using thesingle-letter amino acid code. The numbers at right refer to aminoacids, with methionine (nucleotides 16-18) being number 1. The potentialN-terminal signal sequence (SP) and the transmembrane domain (TMS) areindicated with dashed lines above the sequence, and cysteine residues inthe extracellular domain are circled. A schematic of the predictedprotein structure is indicated below. N-linked glycosylation sites andthe location of the N-terminal peptide sequence (Pep. Seq.) areindicated. The nucleic acid sequence is also identified as SEQ ID:NO:1.

[0032]FIG. 12. Identification of KL-Specific RNA Transcripts in BALB/c3T3 Cell RNA by Northern Blot Analysis. Poly(A)⁺ RNA (4 μg) from BALB/c3T3 cells was electro-phoretically separated, transferred tonitrocellulose, and hybridized with ³²p. labeled 1.4 kb KL cDNA. Themigration of 18S and 285 ribosomal RNSs is indicated.

[0033]FIG. 13. SDS-PAGE Analysis of KL.

[0034] A. Silver staining of KL.

[0035] B. Autoradiography of ^(125I)-KL.

[0036]FIG. 14. Binding of ¹²⁵I-K to Mast Cells and c-kit-Expressing ψ2Cells.

[0037] A. NIH ψ2/c-kit cells containing the pLJ c-kit expression vectorand expressing a high level of high c-kit protein.

[0038] B. Mast cells derived from bone marrow of +/+ or W/W^(v) adultmice or fetal liver cells of W/W or a normal littermate control (W/+ or+/+).

[0039]FIG. 15. Coprecipitation and Cross-Linking of ¹²⁵I-KL with thec-kit receptor on mast cells.

[0040] A. Coprecipitation of KL with normal rabbit serum (NRS) or twoanti-c-kit rabbit antisera (α-c-kit).

[0041] B. Cross-linking of KL to c-kit with disuccinimidyl substrate.SDS-page analysis was on either 12% or 7.5% polyacrylamide gels.Cross-linked species are labeled “KL+cK”.

[0042]FIG. 16. RFLP analysis of Taq1-digested DNA from S1/+ and SIISImice. The S1 allele from C3HeB/Fej a/a CaJ S1 Hm mice was introducedinto a C57BL/6J S1 Hm mice was introduced into a C57BL/6J background,and progeny of a C57BL/6J S1^(C3H)×S1^(C3H) cross were evaluated.

[0043] A. Hybridazation of the 1.4 kB KL cDNA probe to DNA from twononanemic (lanes SII+) and two anemic (lanes SIISI) mice. Nohybridization to the DNA from the SIISI mice was detected.

[0044] B. Hybridization of the same blot to TIS Dra/SaI, a probe that istightly linked to S1 (see Detailed Description, infra). This probeidentifies a 4 kB C3HeB/FeJ-derived allele and a 2 kb C57BL/6J allele inthe SI^(c3H)1S1^(c3H) homozygotes.

[0045]FIG. 17. Nucleotide and predicted amino acid sequence of KL-1,KL-2 and KL-S1^(d) cDNAs. The nucleotide sequence of the KL cDNAobtained from the Balb3T3 cell plasmid cDNA library is shown. The RT-PCTproducts from different tissues and S1^(d)/+ total RNA, KL-1, KL-2 andKL-S1^(d), were subcloned and subjected to sequence analysis. Opentriangles indicate the 5′ and 3′ boundaries of the exon which is splicedout in KL-2: the closed triangles indicate the deletion endpoints in theS1^(d) cDNA. The 67 nucleotide inset sequence of the S1d cDNA is shownabove the KL cDNA sequence. Arrows indicate the putative proteolyticcleavage sites in the extracellular region of KL-1. The signal peptide(SP) and transmembrane segment (TMS) are indicated with overlying lines.

[0046]FIG. 18. Panels A and B. Identification by RT-PCR cloning of KLcDNAs from normal tissues and S1^(d) mutant fibroblasts. Total RNA wasobtained from different tissues of C57BI6/J mice and S1^(d)/+fibroblasts. RT-PCR reactions with RNA (10 μg) from normal tissues andBalb 3T3 cells were done using primers #1 and #2 and reactions with RNAfrom +/+ and S1^(d)/+ fibroblasts were done by using the primercombinations #1, +#2, #1+#3 and #1+#4. The reaction products wereanalyzed by electropnoresis in 1% NuSieve agarose gels in the presenceof 0.25 μg/ml ethidium bromide. The migration of φX174 Hae III DNAmarkers is indicated.

[0047]FIG. 19. Topology of different KL protein products. Shaded areasdelineate N-terminal signal peptides, solid black areas transmembranedomains and Y N-linked glycosylation sites. Dotted lines indicate theexon boundaries of the alternatively spliced exon and correspondingamino acid numbers are indicated. Arrows indicate the presumedproteolytic cleavage sites. The shaded region at the C-terminus ofKL-S1^(d) indicates amino acids that are not encoded by KL. KL-Sdesignates the soluble form of KL produced by proteolytic cleavage orthe C-terminal truncation mutation of KL.

[0048]FIG. 20. Identification of KL-1 and KL-2 transcripts in differenttissues by RNase protection assays. ³²P-labelled antisense riboprobe(625 nt.) was hybridized with 20 μg total cell RNA from tissues andfibroblasts except for lung and heart where 10 μg was used. Upon RNasedigestion, reaction mixtures were analyzed by electrophoresis in a 4%polyacrylamide/urea gel. For KL-1 and KL-2 protected fragments of 575nts. and 449 nts., are obtained respectively. Autoradiographic exposureswere for 48 or 72 hours, except for the 3T3 fibroblast RNA, which wasfor 6 hours.

[0049]FIG. 21. Panels A-C. Biosynthetic characteristics of KL-1 and KL-2protein products in COS cells. COS-1 cells were transfected with 5 μg ofthe KL-1 and KL-2 expression plasmids, using the DEAE-dextran method.After 72 hours the cells were labelled with ³⁵S-Met for 30 minutes andthen chased with complete medium. Supernatants and cell lysates wereimmunoprecipitated with anti-KL rabbit serum. Immunoprecipitates wereanalyzed by SDS-PAGE (12%). Migration of molecular weight markers isindicated in kilo daltons (kD).

[0050]FIG. 22. Panels A-C. PMA induced cleavage of the KL-1 and KL-2protein products. COS-1 cells were transfected with 5 μg of the KL-l andKL-2 expression plasmids and after 72 hours the cells were labelled with³⁵S-Met for 30 minutes and then chased with medium a) in the absence ofserum; b) containing the phorbol ester PMA (1 μM and c) containing thecalcium ionophore A23187 (1 μM). Supernatants and cell lysates wereimmunoprecipitated with anti-KL rabbit serum. Immunoprecipitates wereanalyzed by SDS-PAGE (12%). Migration of molecular weight markers isindicated in kilo daltons (kD).

[0051]FIG. 23. Panels A and B. Biosynthetic characteristics of KL-S1^(d)and KL-S protein products in COS cells.

[0052]FIG. 24. Determination of biological activity in COS cellsupernatants. Supernatants from COS cells transfected with the KL-1,KL-2, KL-S1^(d) and KL-S expression plasmids were assayed for activityin the mast cell proliferation assay. Serial dilutions of supernatantwere incubated with BMMCs and incorporation of ³H-thymidine wasdetermined from 24-30 hours of culture.

[0053]FIG. 25. Synergism between recombinant human (rh) IL-1β (100 U/mL,rmKL (10 to 100 ng/mL), and rhM-CSF, rhG-CSF, and rmIL-3 (all at 1,000U/mL) in the HPP-CFU assay. Four-day post-5-FU murine bone marrow wascultured in 60-mm Petri dishes with a 2 mL 0.5% agarose underlayercontaining cytokines, overlayed with 1 mL of 0.36% agarose containing2.5×10⁴ marrow cells. Following a 12-day incubation under reduced oxygenconditions, cultures were scored from colonies of greater than 0.5 mmdiameter.

[0054]FIG. 26. Secondary CFU-GM or delta assay showing the fold increaseof GM-CSF-responsive CFU-GM in a 7-day suspension culture of 24-hourpost 5-FU murine bone marrow. Marrow cells (⅖×10⁵/mL) were cultured for7 days with the cytokine combinations indicated and recovered cellsreclioned in a GM-CSF-stimulated colony assay. The fold increase is theratio of the number of CFU-GM recovered in the secondary clonogenicassay over the input number of CFU-GM determined in the primaryclonogenic assay over the input number of CFU-GM determined in theprimary clonogenic assay with GM-CSG, rmKL was used as 20 ng.mL, rhIL-6at 50 ng/mL, rhIL-1β at 100 U/mL, and rhGM-CSF or rmIL-3 at 1,000 U/mL.

[0055]FIG. 27. Amplification of hematcpoiesis in cultures of 24 hourspost 5-FU bone marrow cultured for 7 days in suspension in the presenceof IL-1+IL-3 +KL. Cells, 10⁴, (after substraction of granulocytes andlymphocytes) and containing 2.5% HPP-CFU responsive to IL-1+IL-3+KL inprimary clonogenic assay, were incubated in suspension and the totalcells and HPP-CFU responsive to IL-1+IL-3+KL, or CFU-GM responsive tormGM-CSF were determined after 7 days in secondary clonogenic assays.The calculations are based on the ratio of output cells to inputHPP-CFU.

[0056]FIG. 28. The effects of IL-6, IL-1, and KL alone or in combinationon colony growth from normal murine bone marrow. Control cultures weregrown in the absence of any growth factors. The seven combinations orIL-6, IL-1, and KL were tested alone or in combination with the CSF'sG-CSF, M-CSF, GM-CSF, and IL-3. The data are presented as the mean plusthe SE of triplicate cultures.

[0057]FIG. 29. Synergism among IL-6, IL-1 and CSF's in the stimulationof HPP-CFC from 5-FU-purged bone marrow. Bone marrow was harvested 1-7days after the administration of 5-FU (top to bottom) and grown in thepresence of G-CSF, M-CSF, and IL-3+IL-6, IL-1 or IL-6 plus IL-1. Thedata are presented as total CFU-C (HPP-CFC plus LPP-CFC) per 1×10⁵ to1×10⁴ (d1 5-FU to d7 5-FU) bone marrow cells. The data represent the manplus SE of triplicate cultures.

[0058]FIG. 30. KL synergistically stimulates HPP-CFC in combination withother cytokines. As in FIG. 1, 40 combinations of cytokines were testedfor their ability to stimulate CFU-C (HPP-CFC plus LPP-CFC) from B<harvested after 5-FU injection. Colony numbers represent the mean plusSE of triplicate cultures of 1×10⁵ d1 5-FU BM or 1×10⁴ d7 5-FU BM cells.

[0059]FIG. 31. The expansion of total cell numbers in Δ-culturesrequires the combined stimulation of multiple growth factors. Thenumbers of nonadherent cells present in Δ-cultures after 7 days ofgrowth were determined as described in the Materials and methods. Thedashed line represents the 2.5×10⁵ d1 5-FU BM cells used to inoculatethe cultures. The morphologies of the recovered cells are discussed inthe text. The data are presented as the mean plus SE 2-16 experiments.

[0060]FIG. 32. IL-6, IL-1, and KL, alone or in combination, aresynergistic with CSF's in the expansion of LPP-CFC in Δ-cultures. Thefor LPP-CFC grown in the presence of G-CSF, M-CSF, GM-CSF, IL-3 or IL-1plus IL-3 were calculated as described in the Materials and methods. TheΔ-values were calculated from the average of triplicate primary andsecondary colony counts. The results are presented as the mean ±SE of6-11 Δ-values pooled from two or three experiments. Note that theLPP-CFC Δ-values are on a log scale.

[0061]FIG. 33. IL-6, IL-1 and KL alone or in combination, act with CSF'sin the expansion of HPP-CFC in Δ-cultures. All HPP-CFC were grown in thepresence of IL-1 plus IL-3. The Δ-values were calculated from theaverage of triplicate primary and secondary colony counts. The resultsare presented as the mean ±SE of 2-11 experiments. Note that the HPP-CFCΔ-values are on a log scale.

[0062]FIG. 34. Progenitors responsive to IL-1 plus KL are not expandedin Δ-cultures. IL-1 plus IL-3 was compared to IL-1 plus KL foreffectiveness in stimulating primary and secondary HPP-CFC and LPP-CFCin the Δ-assay. The Δ-values were calculated from the average oftriplicate CFU-C assays. The data shown represent the results from oneexperiment. Note that the Δ-values are on a log scale.

[0063]FIG. 35. The numbers CFU-S are expanded in Δ-cultures. TheΔ-values for the expansion of HPP-CFC, LPP-CFC, and CFU-S that occur inthe in vitro Δ-assay or in vivo after 5-FU administration were compared.The Δ-values for the in vivo expansion of progenitor cells were measuredby dividing the numbers of progenitors per femur observed 8 days after5-FU administration by the numbers observed 1 day following 5-FUtreatment. The data represent the mean plus SE of one to threeexperiments.

DETAILED DESCRIPTION OF THE INVENTION

[0064] The relationship of KL to the c-kit receptor has now beendefined, and it is shown that KL is the ligand of c-kit based on bindingand cross-linking experiments. N-terminal protein sequence of KL wasused to derive KL-specific cDNA clones. These cDNA clones were used toinvestigate the relationship of the KL gene to the S1 locus, and it wasdemonstrated that KL is encoded by the S1 locus.

[0065] The hematopoietic growth factor KL was recently purified fromconditioned medium of BALB/c 3T3 fibroblasts, and it has the biologicalproperties expected of the c-kit ligand (37). KL was purified based onits ability to stimulate the proliferation of BMMC from normal mice butnot from W mutant mice in the absence of IL-3. The purified factorstimulates the proliferation of BMMC and CTMC in the absence of IL-3 andtherefore appears to play an important role in mature mast cells. Inregard to the anticipated function of c-kit in erythropoiesis, KL wasshown to facilitate the formation of erythroid bursts (day 7-14 BFU-E)in combination with erythropoietin. The soluble form of KL, which hasbeen isolated from the conditioned medium of Balb/3T3 cells has amolecular mass of 30 kD and a pI of 3.8; it is not a disulfide-linkeddimer, although the characteristics of KL upon gel filtration indicatethe formation of noncovalently linked dimers under physiologicalconditions.

[0066] The predicted amino acid sequence of KL, deduced from the nucleicacid sequence cDNAs, indicates that KL is synthesized as a transmembraneprotein, rather than as a secreted protein. The soluble form of KL thenmay be generated by proteolytic cleavage of the membrane-associated formof KL. The ligand of the CSF-1 receptor, the closest relative of c-kit,shares the topological characteristics of KL and has been shown to beproteolytically cleaved to produce the soluble growth factor (44, 45). Arecent analysis of the presumed structural characteristics of KL,furthermore indicates a relationship of KL and CSF-1 based on amino acidhomology, secondary structure and exon arrangements indicating anevolutionary relationship of the two factors and thus strengthening thenotion that the two receptor systems evolved from each other (4).

[0067] Alternatively spliced KL mRNAs which encode two different formsof the KL protein, i.e., KL-1 and KL-2, have recently been described(15). The KL encoded protein products have been defined andcharacterized in COS cells transfected with the KL cDNAs and extendedthe findings of Flanagan et al. in several ways. As noted hereinabove,KL is synthesized as a transmembrane protein which is proteolyticallycleaved to produce the soluble form of KL. The protein product of thealternatively spliced transcript of KL, KL-2, which lacks the exon thatencodes the presumptive proteolytic cleavage site was shown to displayturnover characteristics that are distinct from those of KL-1. Inaddition, the proteolytic cleavage of both KL-1 and KL-2 can beregulated by agents such as PMA and the calcium ionophore A23187. Therelative abundance of KL-1 and KL-2 has been determined in a widevariety of different mouse tissues. This indicates that the expressionof KL-1 and KL-2 is controlled in a tissue specific manner.

[0068] The gene products of the S1^(d) allele have also been defined(15). S1^(d) results from a deletion within KL which includes thesequences encoding the transmembrane and cytoplasmic domains of theprotein resulting in a biologically active, secreted mutant KL protein.The respective roles of the soluble and cell-associated forms of KL inthe proliferative and migratory functions of c-kit are discussed in thelight of these results.

[0069] This invention provides a purified mammalian proteincorresponding to a ligand for the c-kit which comprises a homodimer oftwo polypeptides, each polypeptide having a molecular weight of about 30kD and an isoelectric point of about 3.8. As used herein, the term“c-kit ligand” is to mean a polypeptide or protein which has also beendefined as stem cell factor, mast cell factor and steel factor. As usedherein, c-kit ligand protein and polypeptide encompasses both naturallyoccurring and recombinant forms, i.e., non-naturally occurring forms ofthe protein and the polypeptide which are sufficiently identically tonaturally occurring c-kit to allow possession of similar biologicalactivity. Examples of such polypeptides includes the polypeptidesdesignated KL-1.4 and S-KL, but are not limited to them. Such proteinand polypeptides include derivatives and analogs. In one embodiment ofthis invention, the purified mammalian protein is a murine protein. Inanother embodiment of this invention, the purified mammalian protein isa human protein.

[0070] Also provided by this invention is a purified mammalian proteincorresponding to a c-kit ligand, wherein the purified protein isglycosolated. However, this invention also encompasses unglycosylatedforms of the protein. This invention also encompasses purified mammalianproteins containing glycosolation sufficiently similar to that ofnaturally occurring purified mammalian protein corresponding to c-kitligand. This protein may be produced by the introduction of a cysteinecross-link between the two homodimer polypeptides described hereinaboveby methods known to those of skill in the art.

[0071] Also provided by this invention is a pharmaceutical compositionwhich comprises an effective amount of the purified mammalian proteincorresponding to c-kit ligand described hereinabove and apharmaceutically acceptable carrier.

[0072] Further provided is a pharmaceutical composition for thetreatment of leucopenia in a mammal comprising an effective amount ofthe above mentioned pharmaceutical composition and an effective amountof a hemopoietic factor, wherein the factor is selected from the groupconsisting of C-CSF, GM-CSF and IL-3, effective to treat leucopenia in amammal.

[0073] Also provided by this invention is a pharmaceutical compositionfor the treatment of anemia in a mammal, which comprises an effectiveamount of the pharmaceutical composition described hereinabove and aneffective amount of EPO (erythropoietin) or IL-3, effective to treatanemia in a mammal. Anemia encompasses, but is not limited to DiamondBlack fan anemia and aplastic anemia. However, for the treatment ofBlack fan anemia and aplastic anemia, a pharmaceutical compositioncomprising an effective amount of the composition described hereinaboveand an effective amount of G-CSF and GM-CSF, effective to treat anemiais preferred. A method of treating anemia in mammals by administering tothe mammals the above composition is further provided by this invention.A pharmaceutical composition effective for enhancing bone marrow duringtransplantation in a mammal which comprises an effective amount of thepharmaceutical composition described hereinabove, and an effectiveamount of IL-1 or IL-6, effective to enhance engraphment of bone marrowduring transplantation in the mammal is also provided. A pharmaceuticalcomposition for enhancing bone marrow recovery in the treatment ofradiation, chemical or chemotherapeutic induced bone marrow, aplasia ormyelosuppression is provided by this inventions which comprises aneffective amount of the pharmaceutical composition described hereinaboveand an effective amount of IL-1, effective to enhance bone marrowrecovery in the mammal. Also provided by this invention is apharmaceutical composition for treating acquired immune deficiencysyndrome (AIDS) in a patient which comprises an effective amount of thepharmaceutical composition described hereinabove and an effective amountof AZT or G-CSF, effective to treat AIDS in the patient.

[0074] A composition for treating nerve damage is provided by thisinvention which comprises an effective amount of the pharmaceuticalcomposition described hereinabove in an amount effective to treat nervedamage in a mammal.

[0075] Also provided is a composition for treating infants exhibitingsymptoms of defective lung development which comprises an effectiveamount of the purified mammalian protein and a pharmaceuticallyacceptable carrier, effective to treat infants exhibiting symptoms ofdefective lunq development.

[0076] Further provided is a composition for the prevention of hair lossin a subject which comprises an effective amount of the purifiedmammalian protein corresponding to c-kit ligand and a pharmaceuticallyacceptable carrier, effective to prevent the loss of hair in thesubject. Also provided by this invention is a pharmaceutical compositionfor inhibiting the loss of pigment in a subject's hair which comprisesan effective amount of the purified mammalian protein corresponding toc-kit ligand and a pharmaceutically acceptable carrier, effective toinhibit the loss of pigment in the subject's hair.

[0077] Methods of treating the above-listed disorders by theadministration of the effective composition, in an amount effective totreat that disorder, also is provided.

[0078] As used herein, the terms “subject” shall mean, but is notlimited to, a mammal, animal, human, mouse or a rat. “Mammal” shallmean, but is not limited to meaning a mouse (murine) or human.

[0079] This invention provides an isolated nucleic acid molecule whichencodes an amino acid sequence corresponding to a c-kit ligand (KL).Examples of such nucleic acids include, but are not limited to thenucleic acids designated KL 1.4, K1-1, KL-2 or S-KL. The invention alsoencompasses nucleic acids molecules which differ from that of thenucleic acid molecule which encode these amino acid sequences, but whichproduce the same phenotypic effect. These altered, but phenotypicallyequivalent nucleic acid molecules are referred to as “equivalent nucleicacids”. And this invention also encompasses nucleic acid moleculescharacterized by changes in non-coding regions that do not alter thephenotype of the polypeptide produced therefrom when compared to thenucleic acid molecule described hereinabove. This invention furtherencompasses nucleic acid molecules which hybridize to the nucleic acidmolecule of the subject invention. As used herein, the term “nucleicacid” encompasses RNA as well as single and double-stranded DNA andcDNA. In addition, as used herein, the term “polypeptide” encompassesany naturally occurring allelic variant thereof as well as man-maderecombinant forms.

[0080] For the purposes of this invention, the c-kit ligand (KL) is ahuman c-kit ligand (KL) or a murine c-kit ligand (KL).

[0081] Also provided by this invention is a vector which comprises thenucleic acid molecule which encodes an amino acid sequence correspondingto a c-kit ligand (KL). This vector may include, but is not limited to aplasmid, viral or cosmid vector.

[0082] This invention also provides the isolated nucleic acid moleculeof this invention operatively linked to a promoter of RNA transcription,as well as other regulatory sequences. As used herein, the term“operatively linked” means positioned in such a manner that the promoterwill direct the transcription of RNA off of the nucleic acid molecule.Examples of such promoters are SP6, T4 and T7. Vectors which containboth a promoter and a cloning site into which an inserted piece of DNAis operatively linked to that promoter are well known in the art.Preferable, these vectors are capable of transcribing RNA in vitro.Examples of such vectors are the pGEM series [Promega Biotec, Madison,Wis.].

[0083] A host vector system for the production of the c-kit ligand (KL)polypeptide is further provided by this invention which comprises one ofthe vectors described hereinabove in a suitable host. For the purposesof this invention, a suitable host may include, but is not limited to aneucaryotic cell, e.g., a mammalian cell, or an insect cell forbaculovirus expression. The suitable host may also comprise a bacteriacell such as E. coli, or a yeast cell.

[0084] To recover the protein when expressed in E. coli, E. coli cellsare transfected with the claimed nucleic acids to express the c-kitligand protein. The E. coli are grown in one (1) liter cultures in twodifferent media, LB or TB and pelleted. Each bacterial pellet ishomogenized using two passages through a French pressure cell at 20′0001b/in² in 20 ml of breaking buffer (below). After a high speed spin 120krpm×20 minutes) the supernatants were transferred into a second tube.The c-kit protein or polypeptide is located in the particulate fraction.This may be solubilized using 6M quanidium-HCI or with 8M urea followedby dialysis or dilution.

[0085] Breaking Buffer

[0086] 50 mM Hepes, pH 8.0

[0087] 20% glycerol

[0088] 150 mM NaCl

[0089] 1 mM Mg So₄

[0090] 2 mM DTT

[0091] 5 mM EGTA

[0092] 20 μg/ml DNAse I.

[0093] A purified soluble c-kit ligand (KL) polypeptide as well as afragment of the purified soluble c-kit ligand (KL) polypeptide isfurther provided by this invention.

[0094] In one embodiment of this invention, the c-kit ligand polypeptidecorresponds to amino acids 1 to 164. In other embodiments of thisinvention, the c-kit ligand polypeptide corresponds to amino acids 1 toabout 148, or fusion polypeptides corresponding to amino acids 1 toabout 148 fused to amino acids from about 165 to about 202 or 205, aswell as a fusion polypeptide corresponding to amino acids 1 to about 164fused to amino acids 177 to about amino acid 202 or about amino acid205.

[0095] In another embodiment of this invention, the c-kit ligandpolypeptide may comprise a polypeptide corresponding to amino acids 1 toabout 164 linked to a biologically active binding site. Such biologicalactive binding sites may comprise, but are not limited to an amino acidscorresponding to an attachment site for binding stromal cells, theextracellular matrix, a heparin binding domain, a hemonectin bindingsite or cell attachment activity. For example, see U.S. Pat. Nos.4,578,079, 4,614,517 and 4,792,525, issued Mar. 25, 1986; Sep. 30, 1986and Dec. 20, 1988, respectively.

[0096] In one embodiment of this invention, the soluble, c-kit ligand(KL) polypeptide is conjugated to an imageable agent. Imageable agentsare well known to those of ordinary skill in the art and may be, but arenot limited to radioisotopes, dyes or enzymes such as peroxidase oralkaline phosphate. Suitable radioisotopes include, but are not limitedto ¹²⁵I, ³²P, and 35S.

[0097] These conjugated polypeptides are useful to detect the presenceof cells, in vitro or in vivo, which express the c-kit receptor protein.When the detection is performed in vitro, a sample of the cell or tissueto be tested is contacted with the conjugated polypeptide under suitableconditions such that the conjugated polypeptide binds to c-Kit receptorpresent on the surface of the cell or tissue; then removing the unboundconjugated polypeptide, and detecting the presence of conjugatedpolypeptide, bound; thereby detecting cells or tissue which express thec-kit receptor protein.

[0098] Alternatively, the conjugated polypeptide may be administered toa patient, for example, by intravenous administration. A sufficientamount of the conjugated polypeptide must be administered, and generallysuch amounts will vary depending upon the size, weight, and othercharacteristics of the patient. Persons skilled in the art will readilybe able to determine such amounts.

[0099] Subsequent to administration, the conjugated polypeptide which isbound to any c-kit receptor present on the surface of cells or tissue isdetected by intracellular imaging.

[0100] In the method of this invention, the intracellular imaging maycomprise any of the numerous methods of imaging, thus, the imaging maycomprise detecting and visualizing radiation emitted by a radioactiveisotope. For example, if the isotope is a radioactive isotope of iodine,e.g., ¹²⁵I, the detecting and visualizing of radiation may be effectedusing a gamma camera to detect gamma radiation emitted by theradioiodine.

[0101] In addition, the soluble, c-kit ligand (KL) polypeptide fragmentmay be conjugated to a therapeutic agent such as toxins,chemotherapeutic agents or radioisotopes. Thus, when administered to apatient in an effective amount, the conjugated molecule acts as a tissuespecific delivery system to deliver the therapeutic agent to the cellexpressing c-kit receptor.

[0102] A method for producing a c-kit ligand (KL) polypeptide is alsoprovided which comprises growing the host vector system describedhereinabove under suitable conditions permitting production of the c-kitligand (KL) polypeptide and recovering the resulting c-kit ligand (KL)polypeptide. This invention also provides the c-kit ligand (KL)polypeptide produced by this method.

[0103] This invention further provides c-kit ligand antagonists. Thesecould be small molecule antagonists found by screening assays on thec-kit receptor. Alternatively, they could be antisense nucleic acidmolecules, DNA, RNA based on ribose or other sugar backbone, withthiophosphate, methyl phosphate, methyl phosphonate linkages between thesugars. These antisense molecules would block the translation of c-kitligand in vivo.

[0104] A soluble, mutated c-kit ligand (KL) antagonist is also provided,wherein this mutated polypeptide retains its ability to bind to thec-kit receptor, but that the biological response which is mediated bythe binding of a functional ligand to the receptor is destroyed. Thus,these mutated c-kit ligand (KL) polypeptides act as antagonists to thebiological function mediated by the ligand to the c-kit receptor byblocking the binding of normal, functioning ligands to the c-kitreceptor. The KL Antagonist may be prepared by random mutagenesis. Amutated or modified KL molecule that was incapable of dimerizing mightbe an effective antagonist. KL shows a great deal of homology withM-CSF, which contains several a-helices which are believed to beimportant for dimerization (102). Site directed mutagenesis in thesehelical regions could block the ability to dimerize. Alternatively, amutated KL could form a heterodimer with normal, functioning KL, but theheterodimer would not be able to activate the c-kit receptor. Becausethe c-kit receptor itself needs to dimerize to be become an activekinase, a soluble, mutated KL that bind to the c-kit receptor yet blocksthe receptor dimerization would be an effective antagonist.

[0105] A pharmaceutical composition which comprises the c-kit ligand(KL) purified by applicants or produced by applicants' recombinantmethods and a pharmaceutically acceptable carrier is further provided.The c-kit ligand may comprise the isolated soluble c-kit ligand of thisinvention, a fragment thereof, or the soluble, mutated c-kit ligand (KL)polypeptide described hereinabove. As used herein, the term“pharmaceutically acceptable carrier” encompasses any of the standardpharmaceutical carriers, such as a phosphate buffered saline solution,water, and emulsions, such as an oil/water or water/oil emulsion, andvarious types of wetting agents. Included in these pharmaceuticalcarriers would be a nebulized aerosol form.

[0106] The KL antagonists described above could be used in a variety oftreatments including asthma, allergies, anaphylaxis, allergic asthma,arthritis including rheumatoid arthritis, papillary conjunctivitis,leukemia, melanoma, dermal allergic reactions, scleroderma.

[0107] This invention further provides a substance capable ofspecifically forming a complex with the c-kit ligand protein, thesoluble, c-kit ligand (KL) polypeptide, or a fragment thereof, describedhereinabove. This invention also provides a substance capable ofspecifically forming a complex with the c-kit ligand (KL) receptorprotein. In one embodiment of this invention, the substance is amonoclonal antibody, e.g., a human monoclonal antibody.

[0108] A method of modifying a biological function associated with c-kitcellular activity is provided by this invention. This method comprisescontacting a sample of the cell, whose function is to be modified, withan effective amount of a pharmaceutical composition describedhereinabove, effective to modify the biological function of the cell.Biological functions which may be modified by the practice of thismethod include, but are not limited to cell-cell interaction,propagation of a cell that expresses c-kit and in vitro fertilization.This method may be practiced in vitro or in vivo. When the method ispracticed in vivo, an effective amount of the pharmaceutical compositiondescribed hereinabove is administered to a patient in an effectiveamount, effective to modify the biological function associated withc-kit function.

[0109] A further aspect of this invention are ex-vivo methods andcompositions containing KL in a suitable carrier for ex-vivo use. Theseaspects include:

[0110] 1. a method for enhancing transfection of early hematopoieticprogenitor cells with a gene by first contacting early hematopoieticcells with the composition containing KL and a hematopoietic factor andthen transfecting the cultured cells of step (a) with the gene.

[0111] 2. a method of transferring a gene to a mammal which comprises a)contacting early hematopoietic progenitor cells with the compositioncontaining KL b) transfecting the cells of (a) with the gene; and c)administering the transfected cells of (b) to the mammal. In thesemethods the gene may be antisense RNA or DNA.

[0112] Compositions containing KL can be used for expansion ofperipheral blood levels ex-vivo and an effective amount of ahematopoietic growth factor or factors. The hematopoietic growth factorIL-1, IL-3, IL-6, G-CSF, GM-CSF or combination thereof are particularlysuited (see FIG. 26). A method for the expansion of peripheral blood isalso provided.

[0113] Methods and compositions containing KL are provided for boostingplatelet levels or other cell types (IL-6 seems particularly suited).

[0114] This invention further provides a method of modifying abiological function associated with c-kit cellular activity bycontacting a cell with KL. The cell may express c-kit or may be ahematopoietic cell or may be involved in vitro fertilization.

[0115] This invention also provides a method of stimulating theproliferation of mast cells in a patient which comprises administeringto the patient the pharmaceutical composition described hereinabove inan amount which is effective to stimulate the proliferation of the mastcells in the patient. Methods of administration are well known to thoseof ordinary skill in the art and include, but are not limited toadministration orally, intravenously or parenterally. Administration ofthe composition will be in such a dosage such that the proliferation ofmast cells is stimulated. Administration may be effected continuously orintermittently such that the amount of the composition in the patient iseffective to stimulate the proliferation of mast cells.

[0116] A method of inducing differentiation of mast cells or erythroidprogenitors in a patient which comprises administering to the patientthe pharmaceutical composition described hereinabove in an amount whichis effective to induce differentiation of the mast cells or erythroidprogenitors is also provided by this invention. Methods ofadministration are well known to those of ordinary skill in the art andinclude, but are not limited to administration orally, intravenously orparenterally. Administration of the composition will be in such a dosagesuch that the differentiation of mast cells or erythroid progenitors isinduced. Administration may be effected continuously or intermittentlysuch that the amount of the composition in the patient is effective toinduce the differentiation of mast cells or erythroid progenitors.

[0117] This invention further provides a method of boosting orstimulating levels of progenitors cells when using c-kit ligand alone orin combination. Particularly effective combinations were with G-CSF,GM-CSF, IL-1, IL-3, IL-6, IL-7 and MIP1α. The combination KL plus IL-1,IL-3 and IL-6 was maximally effective. However, IL-1, IL-3, IL-6 andGM-CSF were moderately effective alone. Particularly as shown in thegrowth of high proliferative potential colony forming assay (HPP-CFU) ofbone treated with 5-fluorouracil (5-FU). Such combinations can be usedin vivo. in vitro and ex-vivo.

[0118] This invention also provides a method of facilitating bone marrowtransplantation or treating leukemia in a patient which comprisesadministering to the patient an effective amount of the pharmaceuticalcomposition described hereinabove in an amount which is effective tofacilitate bone marrow transplantation or treat leukemia. Methods ofadministration are well known to those of ordinary skill in the art andinclude, but are not limited to administration orally, intravenously orparenterally. Administration of the composition will be in such a dosagesuch that bone marrow transplantation is facilitated or such thatleukemia is treated. Administration may be effected continuously orintermittently such that the amount of the composition in the patient iseffective. This method is particularly useful in the treatment of acutemyelogenous leukemia and modifications of chronic myelogenous leukemia.The c-kit ligand would increase the rate of growth of the white bloodcells and thereby make them vulnerable to chemotherapy.

[0119] This invention also provides a method of treating melanoma in apatient which comprises administering to the patient an effective amountof a pharmaceutical composition described hereinabove in an amount whichis effective to treat melanoma. Methods of administration are well knownto those of ordinary skill in the art and include, but are not limitedto administration orally, intravenously or parenterally. Administrationof the composition will be in such a dosage such that melanoma istreated. Administration may be effected continuously or intermittentlysuch that the amount of the composition in the patient is effective.

[0120] The soluble, c-kit ligand (KL) polypeptide may also be mutatedsuch that the biological activity of c-kit is destroyed while retainingits ability to bind to c-kit. Thus, this invention provides a method oftreating allergies in a patient which comprises administering to thepatient an effective amount of the soluble, mutated c-kit liganddescribed hereinabove and a pharmaceutically acceptable carrier, in anamount which effective to treat the allergy. Such a composition could bedelivered in aerosol form with a nebulizing an aqueous form of themutated c-kit ligand antagonist. The KL antagonist described hereinabovewould also be an effective against allergies, once again in aerosolform.

[0121] A topical pharmaceutical composition of the c-kit ligandantagonist would be an effective drug for use with arthritis, rheumatoidarthritis, scleroderma, acute dermal allergic reactions. The c-kitligand antagonist could also be effective against allergicconjunctivitis, post-allergic tissue damage or as a prophylactic againstanaphylactic shock. Because mast cells mediate histamine response, ac-kit antagonist or an antisense molecule complementary to c-kit ligandwould be effective in blocking histamine mediated responses includingallergies and gastric acid secretion.

[0122] The c-kit antagonist would be effective as a treatment ofmelanoma because melanocytes are very dependent on KL for growth. In asimilar manner the KL antagonist could be used against leukemia.

[0123] As is well known to those of ordinary skill in the art, theamount of the composition which is effective to treat the allergy willvary with each patient that is treated and with the allergy beingtreated. Administration may be effected continuously or intermittentlysuch that the amount of the composition in the patient is effective.

[0124] Furthermore, this invention provides a method for measuring thebiological activity of a c-kit (KL) polypeptide which comprisesincubating normal bone-marrow mast cells with a sample of the c-kit (KL)polypeptide which comprises incubating normal bone-marrow mast cellswith sample of the c-kid ligand (KL) polypeptide under suitableconditions such that the proliferation of the normal bone-marrow mastcells are induced; incubating doubly mutant bone-marrow mast cells witha sample of the c-kit ligand (KL) polypeptide under suitable conditions;incubating each of the products thereof with ³H-thymidine; determiningthe amount of thymidine incorporated into the DNA of the normalbone-marrow mast cells and the doubly mutant bone marrow mast cells; andcomparing the amount of incorporation of thymidine into the normalbone-marrow mast cells against the amount of incorporation of thymidineinto doubly mutant bone-marrow mast cells, thereby measuring thebiological activity of c-kit kit ligand (KL) polypeptide.

[0125] Throughout this application, references to specific nucleotidesin DNA molecules are to nucleotides present on the coding strand of theDNA. The following standard abbreviations are used throughout thespecification to indicate specific nucleotides: C-cytosine A-adenosineT-thymidine G-guanosine U-uracil

[0126] Experiment Number 1—Purification of c-kit Ligand

[0127] Experimental Materials

[0128] Mice and Embryo Identification

[0129] WBB6 +/+ and W/W^(v), C57B16 W^(v)/+ and WB W/+ mice wereobtained from the Jackson Laboratory (Bar Harbor, Me.). HeterozygousW⁴¹/+ mice were kindly provided by Dr. J. Barker from the JacksonLaboratory and maintained in applicants' colony by brother sistermating. Livers were removed at day 14-15 of gestation from fetusesderived by mating W/+ animals. W/W fetuses were identified by their palecolor and small liver size relative to other W/+ and +/+ fetuses in thelitter. Their identity was confirmed by analysis of the c-kit protein inmast cells derived from each fetus (38).

[0130] Mast Cell Cultures, Preparation of Peritoneal Mast Cell and FlowCytometry

[0131] Mast cells were grown from bone marrow of adult mice and fetalliver cells of day 14-15 fetuses in RPMI-1640 medium supplemented with10% fetal calf serum (FCS), conditioned medium from WEHI-3B cells,non-essential amino acids, sodium pyruvate, and 2-mercapto-ethanol(RPMI-Complete (C)) (60). Non-adherent cells were harvested, refedweekly and maintained at a cell density less than 7×10⁵ cells/ml. Mastcell content of cultures was determined weekly by staining cytospinpreparations with 1% toluidine blue in methanol. After 4 weeks, culturesroutinely contained greater than 95% mast cells and were used fromproliferation assays. Peritoneal mast cells were obtained from C57B1/6mice by lavage of the peritoneal cavity with 7-10 ml of RPMI-C. Mastcells were purified by density gradient centrifugation on 22%Metrizamide (Nycomed, Oslo, Norway) in PBS without Ca⁺⁺ and Mg⁺⁺,essentially as previously described (61). Mast cells were stained with1% toluidine blue in methanol for 5 minutes and washed for 5 minutes inH₂O, and berberine sulfate by standard procedures (62). Mast cells werelabeled with c-kit specific rabbit antisera which recognizesextracellular determinants of c-kit as previously described and analyzedon a FACSCAN (Becton Dickinson) (38).

[0132] Mast Cell Proliferation Assay

[0133] Mast cells were washed three times in RPMI to remove IL-3 andcultured at a concentration of 5×10⁴ c/ml in RPMI-C in a volume of 0.2ml in 96 well plates with two fold serial dilutions of test samples.Plates were incubated for 24 hours at 37° C., 2.5 μC of ³H-TdR was addedper well and incubation was continued for another 6 hours. Cells wereharvested on glass fiber filters and thymidine incorporation into DNAwas determined.

[0134] Preparation of Fibroblast Conditioned Medium

[0135] Balb/3T3 cells (1) were grown to confluence in Dulbecco'sModified MEM (DME) supplemented with 10% calf serum (CS), penicillin andstreptomycin in roller bottles. Medium was removed and cells washed twotimes with phosphate buffered saline (PBS). DME without CS was added andconditioned medium was collected after three days. Cells were refed withserum containing medium for one to two days, then washed free of serum,and refed with serum free medium and a second batch of conditionedmedium was collected after three days. Conditioned medium (CM) wascentrifuged at 2500 rpm for 15 minutes to remove cells, filtered througha 0.45 u filter and frozen at 4° C. The conditioned medium was thenconcentrated 100-200 fold with a Pellicon ultrafiltration apparatusfollowed by an Amicon stirred cell, both with membranes having a cut offof 10,000 kD.

[0136] Column Chromatography

[0137] Blue Agarose chromatography (BRL, Gaithersburg, Md.) wasperformed by using column with a bed volume of 100 ml equilibrated withPBS. 50-80 ml of FCM concentrate was loaded onto the column and afterequilibration for one hour the flow through which contained the activematerial was collected and concentrated to 15-20 ml in dialysis tubingwith PEG 8000.

[0138] Gel filtration chromatography was performed on a ACA54 Ultrogel(LKB, Rockland, Md.) column (2.6×90 cm) which was equilibrated with PBSand calibrated with molecular weight markers; bovine serum albumin (Mr68,000), chymotrypsinogen (Mr 25,700), and ribonuclease A (Mr 14,300),all obtained from Pharmacia, Piscataway, N.J. The concentrate from theBlue Agarose column was loaded onto the gel filtration column, the flowrate adjusted to 37.5 ml/hour and 7.5 ml fractions collected.

[0139] Anion Exchange and Reverse-Phase HPLC (RP-HPLC)

[0140] High performance liquid chromatography was performed using aWaters HPLC system (W600E Powerline controller, 490E programmablemultiwavelength detector, and 810 Baseline Workstation, Waters, Bedford,Mass.). Active fractions from gel filtration were dialyzed in 0.05 MTris-HCl pH 7.8 and loaded onto a Protein-Pak™ DEAE-5PW HPLC column (7.5mm×7.5 cm, Waters), equilibrated with 0.05 M Tris-HCl pH 7.8. Boundproteins were eluted with a linear gradient from 0 to 0.05 M Tris-HCl pH7.8. Bound proteins were eluted with a linear gradient from 0 to 0.4MNaCl in 0.02 M Tris-HCl pH 7.8. The flow rate was 1 ml/minute and 2 mlfractions were collected.

[0141] RP-HPLC was performed using a semi-preparative and an analyticalsize C₁₈ column from Vydac. For both columns buffer A was 100 mMammonium acetate pH 6.0, and buffer B was 1-propanol. The biologicallyactive fractions from anion exchange were pooled and loaded onto thesemi-preparative C₁₈ column. Bound proteins were eluted with a steepgradient of 0%-23% 1-propanol within the first 10 minutes and 23-33%1-propanol in 70 minutes. The flow rate was adjusted to 2 ml/min and 2ml fractions were collected. Biologically active fractions were pooledand diluted 1:1 with buffer A and loaded on the analytical C₁₈ reversephase column. Proteins were eluted with a steep gradient from 0%-26%1-propanol in 10 minutes and then a shallow gradient from 26%-33%1-propanol in 70 minutes. The flow rate was 1 l/min and 1 ml fractionswere collected. Separation on an analytical C4 reverse phase column wasperformed with a linear gradient of acetonitrile from 0-80% in aqueous0.1% TFA.

[0142] Isolectric Focusing (IEF)

[0143] One ml of partially purified KL was supplemented with 20%glycerol (v/v) and 2% ampholine (v/v) at pH 3.5-10 (LKB, Gaithersburg,Md.). A 5 to 60% glycerol density gradient containing 2% ampholine (pH3.5-10) was loaded onto an IEF column (LKB 8100). The sample was appliedonto the isodense region of the gradient, followed by IEF (2000V, 24 h,4° C.). Five ml fractions were collected and the pH determined in eachfraction. The fractions were dialyzed against RPMI-C and then tested forbiological activity.

[0144] Erythroid Progenitor Assays

[0145] Adult bone marrow, spleen and day 14 fetal liver cells wereplated at 10⁵, 10⁶, and 10⁷ cells/ml, respectively, in Iscove's modifiedDulbecco's medium with 1.2% methyl-cellulose, 30% FCS, 100 uM2-mercaptoethanol, human recombinant erythropoietin (2 units/ml, Amgen,Thousand Oaks, Calif.) (Iscove, 1978; Nocka and Pelus, 1987). Cultureswere incubated for 7 days at 37° C. and hemoglobinized colonies andbursts scored under an inverted microscope. 0.1 mM hemin (Kodak) wasadded to cultures of bone marrow cells for optimum growth. Purified KL,IL-3 either as WEHI-3 CM (10%, vol/vol) or recombinant murine IL-3 (50u/ml, Genzyme, Cambridge) was added where indicated.

[0146] Experimental Methods

[0147] Short Term Mast Cell Proliferation Assay Detects a FibroblastDerived Activity

[0148] In order to identify and measure a fibroblast derived growthfactor activity which facilitates the proliferation of normal but notW/W^(v) mast cells, BMMC were washed free of IL-3 containing medium,incubated with medium containing 20 fold concentrated fibroblastconditioned medium (FCM) or WEHI-3 CM (IL-3) and after 24 hours ofincubation ³H-thymidine incorporation was determined. The response ofBMMC derived from normal +/+ and mutant W/W^(v) mice to IL-3 was similar(FIG. 1); in contrast, 20 fold concentrated fibroblast conditionedmedium facilitated the proliferation of +/+ mast cells, but littleproliferation was seen with W/W^(v) mast cells. Concentrated FCM wasalso tested for its ability to stimulate the proliferation of other IL-3dependent cells. The myeloid 32D cells are known to lack c-kit geneproducts (35). No proliferation of the 32D cells was observed with FCM,although normal proliferation was obtained with WEHI-3 CM (not shown).Taken together these results and the known defects in c-kit for both theW and W^(v) alleles (38), suggested that FCM activity was dependent onthe expression of a functional c-kit protein in mast cells (BMMC) andtherefore might be the ligand of the c-kit receptor. In addition the FCMactivity was distinct from IL-3. Therefore, normal and W mutant mastcells provide a simple, specific assay system for the purification ofthe putative c-kit ligand (KL) from fibroblast conditioned medium.

[0149] Purification of the Mast Cell Stimulating Activity KL

[0150] To purify KL, five liters of serum free conditioned medium fromBalb/3T3 fibroblasts was concentrated 50 fold by ultrafiltration. Theconcentrate was passed through a Blue Agarose column equilibrated withPBS and the flow through, which contained the mast cell stimulatingactivity, was collected and concentrated with polyethylene glycol. Inaddition to the determination of the bio-activity by using normal mastcells, peak fractions throughout the purification were also tested withW/W^(v) mast cells where little activity was observed. The material fromthe Blue Agarose column was fractionated by gel filtration using a ACA54 column (FIG. 2A). The biological activity eluted as a major and aminor peak corresponding to 55-70 kD and 30 kD, respectively. Thefractions of the main peak were pooled, dialyzed and fractionated byFPLC chromatography on a DEAE-5PW column with a NaCl gradient (FIG. 2B).The activity eluted at 0.11 M NaCl from the FPLC column. Peak fractionswere pooled and subjected to HPLC chromatography with a semi-preparativeC18 column and an ammonium acetate/n-propanol gradient (FIG. 2C). Theactive material eluted at 30% n-propanol from the semi-preparative C18column was diluted 1:1 with buffer A and rechromatographed by using ananalytical C18 column (FIG. 2D). A single peak of activity eluted againat 30% n-propanol which corresponded to a major peak of absorbance (280nm) in the eluant profile. Similar results were obtained by using a C4column with H₂O and acetonitrile containing 0.1% TFA as solvents (FIG.3B). SDS-PAGE analysis of the active fractions from the separations withboth solvent systems and silver staining revealed one major band with amobility corresponding to a molecular mass of 28-30 kD. The presence andmagnitude of this band correlated well with the peak of biologicalactivity (FIG. 3). There was no significant difference in the migrationof this band under reduced and non-reduced conditions, indicating thatKL was not a disulfide linked dimer (FIG. 3C). Three discrete specieswere observed on both reduced and non-reduced SDS-PAGE indicating sizeheterogeneity of the purified material. The total amount of proteinestimated by absorbance at 280 nm correlated with the amount detected bysilver stain relative to BSA as a reference standard. As indicated inTable 1, the purification of KL from conditioned medium of Balb/3T3cells was more than 3000 fold and the recovery of the initial totalactivity 47%. Half maximal proliferation of +/+ mast cells inapplicants' assay volume of 0.2 ml is defined as 50 units of activityand corresponds to approximately 0.5 ng of protein. Isoelectric focusingof partially purified material (after ion exchange) revealed a majorpeak of activity in the pH range of 3.7-3.9 indicating an isoelectricpoint for KL of 3.7-3.9. TABLE 1 Purification of KL from Balb/3T3Conditioned Medium Total Total Specific Purifi- Purification ProteinActivity Activity cation Yield Step (mg) (U × 10⁻⁵) (U/mg) (Fold) (%)FCM (5L), 152 — — — — 50X Concentrated Blue Agarose 32 720 2.2 × 10⁴ 1100  Gel Filtration 28 480 1.7 × 10⁴ .77 67 DEAE-5PW 3 720 2.4 × 10⁵ 11100  C18-Semiprep .079 600 7.6 × 10⁶ 345 83 C18-Analytical .004 340 8.5× 10⁷ 3863 47

[0151] Proliferative Response to KL of Mast Cells with Different c-kit/WMutations

[0152] Purified KL was tested for its ability to stimulate theproliferation of mast cells derived from wildtype animals as well ashomozygotes and heterozygotes of W, W^(v) , and W⁴¹ alleles. Theoriginal W allele specifies a nonfunctional c-kit receptor and animalshomozygous for the w allele die perinatally, are severely anemic andmast cells derived from W/W fetuses do not proliferate when co-culturedwith Balb/3T3 fibroblasts (63, 38). The W^(v) and W⁴¹ alleles bothspecify a partially defective c-kit receptor and homozygous mutantanimals are viable (64, 65, 38). Homozygous W^(v) animals have severemacrocytic anemia and their mast cells display a minor response in theco-culture assay, and homozygotes for the less severe W⁴¹ allele have amoderate anemia and their mast cells show an intermediate response inthe co-culture assay. Homozygous and heterozygous mutant and +/+ mastcells were derived from the bone marrow for the W and W⁴¹ alleles andfrom day 14 fetal livers for the W allele as described previously (38).Fetal liver derived W/W mast cells did not proliferate in response to KLwhereas both heterozygous (W/+) and normal (+/+) mast cells displayed asimilar proliferative response to KL (FIG. 4). Bone marrow derived mastcells from W^(v)/W^(v) mice were severely defective in their response toKL, although some proliferation, 10% of +/+ values, was observed at 100U/ml (FIG. 4). W^(v)/+ mast cells in contrast to heterozygous WI+mastcells showed an intermediate response (40%) in agreement with thedominant characteristics of this mutation. W⁴¹/W⁴¹ and W⁴¹/+ mast cellswere also defective in their ability to proliferate with KL, althoughless pronounced than mast carrying the W and the W^(v) alleles, which isconsistent with the in vivo phenotype of this mutation (FIG. 4). Theseresults indicate a correlation of the responsiveness of mast carryingthe W, W^(v) and W⁴¹ alleles to KL with the severity and in vivocharacteristics of these mutations. In contrast, the proliferativeresponse of mutant mast cells to WEHI-3CM (IL-3) was not affected by thedifferent W mutations.

[0153] KL Stimulates the Proliferation of Peritoneal Mast Cells

[0154] Mast cells of the peritoneal cavity (PMC) have been wellcharacterized and in contrast to BMMC represent connective tissue-typemast cells (66). PMC do not proliferate in response to IL-3 alone;however, their mature phenotype and viability can be maintained byco-culture with NIH/3T3 fibroblasts (67). Thus, it was of interest todetermine whether KL could stimulate the proliferation of PMC. First,c-kit was examined to determine if it is expressed in PMC. Peritonealmast cells were purified by sedimentation in a metrizamide gradient andc-kit expression on the cell surface analyzed by immunofluorescence withanti-c-kit sera or normal rabbit sera. The PMC preparation was 90-98%pure based on staining with toluidine blue and berberine sulfate.Berberine sulfate stains heparin proteoglycans in granules of connectivetissue mast cells and in addition the dye is also known to stain DNA(FIG. 5) (62). BMMC and mucosal mast cells contain predominantlychondroitin sulfate di-B/E proteoglycans rather than heparinproteoglycans (67); berberine sulfate therefore did not stain thegranules in BMMC (FIG. 5A). Analysis of c-kit expression byflow-cytometry indicated that virtually all PMC expressed c-kit atlevels similar to those observed in BMMC (FIG. 5B). KL was then examinedto determine if it would effect the survival or stimulate theproliferation of PMC (FIG. 5C). Culture of PMC in medium alone, or bythe addition of WEHI-3CM at concentrations optimal for BMMC, results inloss of viability of PMC within 3-4 days although a few cells survivedin WEHI-3CM for longer periods. Culture of PMC in the presence of KLsustained their viability and after two weeks the cell number hadincreased from 5000 to 60,000. A similar increase in the number of BMMCwas observed in response to KL. In contrast to the lack of aproliferative response of PMC to WEHI-3CM, BMMC's proliferated withWEHI-3CM as expected. After one and two weeks in culture, cells werestained with toluidine blue and berberine sulfate. The mature phenotypeof PMC was maintained in culture with 100% of cells staining with bothdyes, although the staining with berberine sulfate was somewhatdiminished when compared with freshly isolated PMC.

[0155] KL Stimulates the Formation of Erythroid Bursts (BFU-E)

[0156] An important aspect of W mutations is their effect on theerythroid cell lineage. The in vivo consequences of this defect aremacrocytic anemia which is lethal for homozygotes of the most severealleles (47, 65). Analysis of erythroid progenitor populations in thebone marrow of W/W^(v) mice indicates a slight decrease of BFU-E andCFU-E (68,69). In livers of W/W fetuses the number of BFU-E is notaffected but a large decrease in the number of CFU-E is seen suggestinga role for c-kit at distinct stages of erythroid maturation presumablyprior to the CFU-E stage (35). In order to evaluate a role for KL inerythropoiesis and to further define its relationship to the c-kitreceptor, the effect of KL on BFU-E formation was determined. Bonemarrow, spleen and fetal liver cells were plated, by using standardculture conditions, in the presence and absence of KL, erythropoietinand WEHI-3 CM. BFU-E were then scored on day 7 of culture. In theabsence of erythropoietin, no erythroid growth was observed with eitherWEHI-3 CM or KL. In the presence of erythropoietin, BFU-E from spleencells were stimulated by KL in a dose dependent manner, from 12BFU-E/10⁶ cells with erythropoietin alone to 50 BFU-E/10⁶ cells withmaximal stimulation at 2.5 ng of KL/ml (FIG. 6). In addition to theeffect on the number of BFU-E, the average size of the bursts wasdramatically increased by KL. THe number of BFU-E obtained by usingspleen cells with KL+erythropoietin was similar to the number observedwith WEHI-3 CM+erythropoietin. In contrast, KL+erythropoietin did notstimulate the proliferation of BFU-E from bone marrow cells, whereasWEHI-3 CM+erythropoietin induced the formation of 18 BFU-E from 10⁵ bonemarrow cells. The effect of KL on day 14 fetal liver cells was alsoexamined and similar results were observed as with spleen cells. Asignificant number of BFU-E from fetal liver cells were observed witherythropoietin alone; however, this number increased from 6±2 to 20±5with 2.5 ng/ml of KL. In the presence of WEHI−3 CM+erythropoietin 18±3BFU-E were observed with fetal liver cells.

[0157] To further evaluate the relationship of KL to c-kit in theerythroid lineage, it was assessed whether KL facilitates the formationof erythroid bursts (BFU-E) from fetal liver cells of W/W mice. W/W andW/+ or +/+ liver cells were prepared from fetuses at day 16.5 ofgestation from mating w/+ mice. The total number of nucleated cells wasreduced eight fold in the liver of the W/W mutant embryo as compared tothe healthy fetuses. The number of BFU-E from W/W and W/+ or +/+ fetalliver was similar in cultures grown with IL-3+ erythropoietin and thelow level of BFU-E in cultures grown with erythropoietin alone wascomparable as well (FIG. 7). KL did not stimulate BFU-E above levelsseen with erythropoietin alone for W/W fetal liver cells, whereas as thenumber of KL dependent BFU-E from W/+ or +/+ liver cells were similar tothose obtained with erythropoietin +IL-3. This result suggests thatresponsiveness of erythroid progenitors to KL is dependent on c-kitfunction.

[0158] Binding Studies with Purified KL

[0159] Purified KL was labelled with ¹²⁵I by the chloramine T method toa high specific activity, i.e., to 2.8×10⁵ cpm/ng. Using the labelledKL, specific binding of KL to mast cells was detected. However, with W/Wmast cells, no binding was detected and good binding to mast cells oflittermates was seen. After binding to mast cells, KL coprecipitatedwith antisera to c-kit. In addition, binding of KL to W mutant mastcells correlates with c-kis expression on the cell surface, V, 37(+)versus W(−).

[0160] Determination of the Peptide Sequence of the c-kit Ligand

[0161] The c-kit receptor protein was isolated as described hereinaboveand the sequence of the protein was determined by methods well known tothose of ordinary skill in the art.

[0162] The single letter amino acid sequence of the protein from theN-terminal is: K E I X G N P V T D N V K D I T K L V A N L P N D Y M I TL N Y V A G M X V L P,

[0163] with:

[0164] K=lysine; E=glutamic acid; I=isoleucine; X=unknown; G=glycine;N=asparagine; P=proline; V=valine; T=threonine; D=aspartic acid;L=leucine; A=alanine; Y=tyrosine; and M=methionine.

[0165] Experimental Discussion

[0166] The finding that the W locus and the c-kit proto-oncogene areallelic revealed important information about the function of c-kit indevelopmental processes and in the adult animal. The knowledge of thefunction of the c-kit receptor in return provided important clues abouttissues and cell types which produce the ligand of the c-kit receptor.In an attempt to identify the c-kit ligand, a growth factor waspurified, designated KL, from conditioned medium of Balb/3T3fibroblasts, a cell type suspected to produce the c-kit ligand, whichhas biological properties expected of the c-kit ligand with regard tomast cell biology and erythropoiesis. KL has a molecular mass of 30 kDand an isoelectric point of 3.8. KL is not a disulfide linked dimer, incontrast to CSF-1, PDGF-A and PDGF-B which have this property (70, 71).Although, the behavior of KL upon gel filtration in PBS indicated a sizeof 55-70 kD which is consistent with the presence of non-covalentlylinked dimers under physiological conditions. KL is different from otherhematopoietic growth factors with effects on mast cells, such as IL-3and IL-4, based on its ability to stimulate the proliferation of BMMCand purified peritoneal mast cells (CTMC), but not BMMCs from W mutantmice. Balb/3T3 fibroblasts are a source for the hematopoietic growthfactors G-CSF, GM-CSF, CSF-1, LIF and IL-6; however, none of these havethe biological activities of KL (35, 71). Furthermore, preliminaryresults from the determination of the protein sequence of KL indicatethat KL is different from the known protein sequences.

[0167] An essential role for c-kit and its ligand in the proliferation,differentiation, and/or survival of mast cells in vivo has been inferredbecause of the absence of mast cells in W mutant mice (72, 73). Theprecise stage(s) at which c-kit function is required in mast celldifferentiation are not known. Mast cells derived in vitro from bonemarrow, fetal liver, or spleen with IL-3 resemble mucosal mast cells(MMC), although they may represent a precursor of both types ofterminally differentiated mast cells, MMC and CTMC (66). Apparently,c-kit is not required for the generation of BMMC from hematopoieticprecursors since IL-3 dependent mast cells can be generated withcomparable efficiency from bone marrow or fetal liver of both normal andW mutant mice (60). The demonstration of c-kit expression in BMMC andCTMC/PMC and the corresponding responsiveness of BMMC and matureCTMC/PMC to KL suggests a role for c-kit at multiple stages in mast celldifferentiation. In addition to fibroblasts, it has been shown that thecombination of IL-3 and IL-4, IL-3 and PMA, or crosslinking of IgEreceptors can stimulate the proliferation of CTMC in vitro (74, 75, 76,77, 78). In contrast to these biological response modifiers, which aremediators of allergic and inflammatory responses, KL by itself in thepresence of FCS is capable of stimulating CTMC proliferation. Therefore,KL may have a mast cell proliferation and differentiation activity whichis independent from these immune responses for its production and actionon target cells.

[0168] The defect W mutations exert on erythropoiesis indicates anessential role for c-it in the maturation of erythroid cells (80, 68,69). The analysis of erythroid progenitors in fetal livers of W/Wfetuses compared with normal littermates suggested that in the absencec-kit function, maturation proceeds normally to the BFU-E stage, butthat progression to the CFU-E stage is suppressed (35). In vitro, thisdefect can be overcome by the inclusion of IL-3 in the culture system,which together with erythropoietin is sufficient to facilitate thematuration of BFU-E from W/W^(v) and +/+ bone marrow (78). in vivo, arole for IL-3 in this process is not known and therefore c-kit may servea critical function in the progression through this stage of erythroiddifferentiation. The ability of KL to stimulate the formation oferythroid bursts from spleen and fetal liver cells together witherythropoietin is consistent with c-kit functioning at this stage oferythroid differentiation. Furthermore, the ability of KL to stimulateW/W BFU-E suggest that c-kit function is required for KL mediated BFU-Eformation and this is similar to the requirement of c-kit function forKL mediated mast cell proliferation. A burst promoting effect ofBalb/3T3 cells on the differentiation of BFU-E from fetal liver cellshad been described previously (79). It is likely that KL is responsiblefor the burst promoting activity of Balb/3T3 cells. An interestingfinding of this study is the inability of KL to stimulate day 7 BFU-Efrom bone marrow cells. This result suggests that BFU-E in fetal liver,adult spleen and adult bone marrow differ in their growth requirements.Recent experiments indicate that KL may stimulate an earliererythroid-multipotential precursor in bone marrow which appears at latertimes in culture (day 14-20). To demonstrate a direct effect of KL onBFU-E formation and to rule out the involvement of accessory cells orother endogenous growth factors, experiments with purified progenitorpopulations need to be performed.

[0169] In addition to the defects in erythropoiesis and mast celldevelopment, W mutations are thought to affect the stem cell compartmentof the hematopoietic system. The affected populations may include thespleen colony forming units (CFU-S) which produce myeloid colonies inthe spleen of lethally irradiated mice as well as cell with long termrepopulation potential for the various cell lineages (81, 46, 47, 81,82). It will now be of interest to determine if there is an effect of KLin the self-renewal or the differentiation potential of hematopoieticstem cell populations, possibly in combination with other hematopoieticgrowth factors, in order to identify the stage(s) where the c-kit/W geneproduct functions in the stem cell compartment.

[0170] Mutations at the steel locus (S1) of the mouse producepleiotropic phenotypes in hematopoiesis, melanogenesis and gametogenesissimilar to those of mice carrying W mutations (47, 51). However, incontrast to W mutations, S1 mutations affect the microenvironment of thecellular target of the mutation and are not cell autonomous (46).Because of the parallel and complementary effects of the W and the S1mutations, it has been suggested that the S1 gene encode the ligand ofthe c-kit receptor or a gene product that is intimately linked to theproduction and/or function of this ligand (9). In agreement with thisconjecture S1/S1^(d) embryo fibroblasts or conditioned medium fromS1/S1^(d) fibroblasts fail to support the proliferation of BMMC and mastcell progenitors, respectively, and presumably do not produce functionalKL (16, 84). If KL is the ligand of the c-kit receptor, then molecularanalysis will enable the determination of the identity of KL with thegene product of the S1 locus; in addition, one would predict thatadministration of KL to mice carrying S1 mutations would lead to thecure of at least some symptoms of this mutation.

[0171] The 1.4 kb cDNA clone is used to screen a human fibroblast or ahuman placenta library using the methods disclosed hereinabove. Uponisolating the gene which encodes the human c-kit ligand, the gene willbe characterized using the methods disclosed hereinabove.

[0172] Experiment Number 2—Isolation of the Nucleic Acid Sequence

[0173] Experimental Materials

[0174] Mice and Tissue Culture

[0175] WBB6+/+, C57BL/6J, C57BL/67 W^(v)/+, WB6W/+, C3HeB/FeJ a/a Ca^(d)S1 Hm, and M. spretus mice were obtained from The Jackson Laboratory(Bar Harbor, Me.). For the interspecific cross, female C57B1/6J and maleM. spretus mice were mated; progeny of this cross were scored forinheritance of C57BL/6J or M. spretus alleles as described infra.(C57BL/6J×M. spretus) F1 female offspring were backcrossed with C57BL/6Jmales.

[0176] Mast cells were grown from the bone marrow of adult +/+,W^(v)/W^(v) and W/+ mice and W/W fetal liver of day 14-15 fetuses inRPMI 1640 medium supplemented with 10% fetal cell serum (FCS),conditioned medium from WEHI-3B cells, nonessential amino acids, sodiumpyruvate, and 2-mercaptoethanol (RPMI-Complete) (36,60). BALB/c 3T3cells (1) were obtained from Paul O'Donnell (Sloan-Kettering Institute,New York, N.Y. ) and were grown in Dulbecco's modified MEM supplementedwith 10% calf serum, penicillin, and streptomycin.

[0177] Purification and Amino Acid Sequence Determination of KL

[0178] KL was purified from conditioned medium of BALB/c 3T3 cells byusing a mast cell proliferation assay as described elsewhere (37).Conditioned medium was then concentrated 100- to 200-fold with aPellicon ultrafiltration apparatus followed by an Amicon stirred cell.The concentrate was then chromatographed on Blue Agarose (BethesdaResearch Laboratories, Gaithersburg, Md.), and the flow-through, whichcontained the active material, was concentrated in dialysis tubing withpolyethylene glycol 8000 and then fractionated by gel filtrationchromatography on an ACA54 Ultrogel (LKB, Rockland, Md.) column. Thebiological activity eluted as a major and a minor peak, corresponding to55-70 kd and 30 kd, respectively. The fractions of the main peak werepooled, dialyzed, and fractionated by FPLC on a DEAE-5PW column with anNaCl gradient. The activity eluted at 0.11 M NaCl from the FPLC column.Peak fractions were pooled and subjected to HPLC with a semi-preparativeC18 column and an ammonium acetate-n-propanol gradient. The activematerial eluted at 30% n-propanol from the semipreparative C18 columnwas diluted 1:1 and re-chromatographed by using an analytical C18column. A single peak of activity eluted again at 30% n-propanol, whichcorresponded to a major peak of absorbance (280 nm) in the eluantprofile. Similar results were obtained by using a C4 column with H₂O andacetonitrile containing 0.1% TFA as solvents. N-terminal amino acidsequence was determined on an Applied Biosystems 477A on-line PTH aminoacid analyzer (Hewick et al., 1961).

[0179] Iodination

[0180] KL was iodinated with chloramine T with modifications of themethod of Stanley and Gilbert (1981). Briefly, the labeling reactioncontained 200 ng of KL, 2 nmol of chloramine T, 10% dimethyl sulfoxide,and 0.02% polyethylene glycol 8000, in a total volume of 25 μl in 0.25 Mphosphate buffer (pH 6.5). The reaction was carried out for 2 min. at 4°C. and stopped by the addition of 2 nmol of cysteine and 4 μM KI. KL wasthen separated from free NaI by gel filtration on a PD10 column(Pharmacia). Iodinated KL was stored for up to 2 weeks at 4° C.

[0181] Binding Assay

[0182] Binding buffer contained RPMI 1640 medium, 5% BSA (Sigma), 20 mMHEPES (pH 7.5) and NaN₃. Binding experiments with nonadherent cells werecarried out in 96-well tissue culture dishes with 2×10⁵ cells per wellin a volume of 100 μl. Binding experiments with ψ2 cells were carriedout in 24-well dishes in a volume of 300 μl. Cells were equilibrated inbinding buffer 15 minutes prior to the addition of competitor or labeledKL. To determine nonspecific binding, unlabeled KL or anti-c-kit rabbitserum was added in a 10-fold excess 30 minutes prior to the addition of¹²⁵I-KL. Cells were incubated with ¹²⁵1I-KL for 90 minutes, andnonadherent cells were pelleted through 150 μl of FCS. Cell pellets werefrozen and counted.

[0183] Immunoprecipitation and Cross-Linking

[0184] BMMC were incubated with ¹²⁵I-KL under standard bindingconditions and washed in FCS and then in PBS at 4° C. Cells were lysedas previously described (35) in 1% Triton X-100, 20 mM Tris (pH 7.4),150 mM NaCl, 20 mM EDTA, 10% glycerol, and protease inhibitorsphenylmethylsufonyl fluoride (1 mM) and leupeptin (20 μg/ml). Lysateswere immunoprecipitated with normal rabbit serum, or c-kit specific seraraised by immunization of rabbits with a fragment of the v-kit tyrosinekinase domain (23); or the murine c-kit expressed from a cDNA in arecombinant vaccinia virus (36). For coprecipitation experiments,immunoprecipitates were washed three times with wash A (0.1% TritonX-100, 20 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol), solubilized inSDS sample buffer, and analyzed by SDS-PAGE and autoradiography. Forcross-linking experiments, cells were incubated with disuccinimidylsubstrate (0.25 mg/ml) in PBS for 30 minutes at 4° C., washed in PBS,and lysed as described above. Washing conditions following precipitationwere as follows: one time in wash B (50 mM Tris, 500 mM NaCl, 5 mM EDTA,0.2% Triton X-100), three times in wash C (50 mM Tris, 150 mM NaCl, 0.1%Triton X-100, 0.1% SDS, 5 mM EDTA), and one time in wash D (10 mM T-is,0.1% Triton X-100).

[0185] cDNA synthesis. PCR Amplification (RT-PCR) and SequenceDetermination

[0186] The RT-PCR amplification was carried out essentially as described(53). For cDNA synthesis, 1 μg of poly(A)⁻RNA from confluent BALB/c 3T3cells in 25 μl of 0.05 M Tris-HCl (pH 8.3), 0.075 M KCl, 3 mM MgCl₂, 10mM dithiothreitol, 200 μM dNTPs and 25 U of RNAsin (Promega) wasincubated with 50 pmol of antisense primer and 50 U of Moloney murineleukemia virus reverse transcriptase at 40° C. for 30 minutes. Another50 U of reverse transcriptase was added, and incubation was continuedfor another 30 minutes. The cDNA was amplified by bringing up thereaction volume to 50 μl with 25 μl of 50 mM KCl, 10 mM Tris-HCl (pH8.3), 1.5 mM MgCl₂, 0.01% (w/v) gelatin, and 200 μM dNTPs, adding 50pmol of sense primer and 2.5 U of Taq DNA polymerase, and amplifying for25-30 cycles in an automated thermal cycler (Perkin-Elmer Cetus). Theamplified fragments were purified by agarose gel electrophoresis,digested with the appropriate restriction enzymes, and subcloned intoM13mp18and M13mp19 for sequence analysis (49).

[0187] cDNA Isolation and Sequencing

[0188] A mouse 3T3 fibroblast lambda g11 cDNA library obtained fromClontech was used in this work. Screening in duplicate was done withEscherichia coli Y1090 as a host bacterium (48); 5′ end-labeledoligonucleotide was used as a probe. Hybridization was in 6× SSC at 63°C., and the final wash of the filters was in 2× SSC, 0.2% SDS at 63° C.Recombinant phage were digested with EcoRI and the inserts subclonedinto M13 for sequence analysis. The nucleotide sequence of these cDNAswas determined, on both strands and with overlaps, by the dideoxy chaintermination method of Sanger et al. (49) by using syntheticoligodeoxynucleotides (17-mers) as primers.

[0189] DNA and RNA Analysis

[0190] Genomic DNA was prepared from tail fragments, digested withrestriction enzymes, electrophoretically fractionated, and transferredto nylon membranes. For hybridization, the 1.4 kb KL cDNA and TISDra/SaI (a probe derived from the transgene insertion site in thetransgenic line TG.EB (85) were used as probes.

[0191] BALB/c 3T3 cells were homogenized in guanidiniu=isothiocyanate,and RNA was isolated according the method of Chirgwin et al. (10). Totalcellular RNA (10 μg) and poly(A)⁺ RNA were fractionated in 1%agarose-formaldehyde gels and transferred to nylon membranes (Nytran,Schleicher & Schuell); prehybridization and hybridization were performedas previously described (86, 35). The 1.4 kb KL cDNA labeled with[³²P]phosphate was used as a probe for hybridization (87).

[0192] Preparation of c-kit and c-kit Ligand Monoclonal antibodies

[0193] For the isolation of human monoclonal antibodies, eight week oldBalb/c mice are injected intraperitoneally with 50 micrograms of apurified human soluble c-kit ligand (KL) polypeptide, or a solublefragment thereof, of the present invention (prepared as described above)in complete Freund's adjuvant, 1:1 by volume. Mice are then boosted, atmonthly intervals, with the soluble ligand polypeptide or soluble ligandpolypeptide fragment, mixed with incomplete Freund's adjuvant, and bledthrough the tail vein. On days 4, 3, and 2 prior to fusion, mice areboosted intravenously with 50 micrograms of polypeptide or fragment insaline. Splenocytes are then fused with non-secreting myeloma cellsaccording to procedures which have been described and are known in theart to which this invention pertains. TWO weeks later, hybridomasupernatants are screened for binding activity against c-kit receptorprotein as described hereinabove. Positive clones are then isolated andpropagated.

[0194] Alternatively, to produce the monoclonal antibodies against thec-kit receptor, the above method is followed except that the method isfollowed with the injection and boosting of the mice with c-kit receptorprotein. Alternatively, for the isolation of murine monoclonalantibodies, Sprague-Dawley rats or Louis rats are injected with murinederived polypeptide and the resulting splenocydes are fused to ratmyeloma (y3-Ag 1.2.3) cells.

[0195] Experimental Results

[0196] Isolation and Characterization of Murine cDNAs Encoding theHematopoietic Growth Factor KL

[0197] The KL protein was purified from conditioned medium from BALB/c3T3 cells by a series of chromatographic steps including anion exchangeand reverse-phase HPLC as described hereinabove (37). As previouslynoted, the sequence of the N-terminal 40 amino acids of KL wasdetermined to be: K E I X G N P V T D N V K D I T K L V A N L P N D Y MI T L N Y V A G M X V L P.

[0198] To derive a nondegenerate homologous hybridization probe, fullydegenerate oligonucleotide primers corresponding to amino acids 10-16(sense primer) and 31-36 (antisense primer) provided with endonucleaserecognition sequences at their 5′ ends were synthesized as indicated inFIG. 8. A cDNA corresponding to the KL mRNA sequences that specify aminoacids 10-36 of KL was obtained by using the reverse transcriptasemodification of the polymerase chain reaction (RT-PCR). Poly (A)⁺ RNAfrom BALB/c 3T3 cells was used as template for cDNA synthesis and PCRamplification in combination with the degenerate oligonucleotideprimers.

[0199] The amplified DNA fragment was subcloned into M13, and thesequences for three inserts were determined. The sequence in between theprimers was found to be unique and to specify the correct amino acidsequence (FIG. 8). An oligonucleotide (49 nucleotides) corresponding tothe unique sequence of the PCR products was then used to screen a λ gt11mouse fibroblast library. A 1.4 kb clone was obtained that, in its 3′half, specifies an open reading frame that extends to the 3′ end of theclone and encodes 270 amino acids (FIG. 11). The first 25 amino acids ofthe KL amino acid sequence have the characteristics of a signalsequence. The N-terminal peptide sequence that had been derived from thepurified protein (amino acids 26-65) follows the signal sequence. Ahydrophobic sequence of 21 amino acids (residues 217-237) followed atits carboxyl end by positively charged amino acids has the features of atransmembrane segment. In the sequence between the signal peptide andthe transmembrane domain, four potential N-linked glycosylation sitesand four irregularly spaced cysteines are found. A C-terminal segment of33 amino acids follows the transmembrane segment without reaching atermination signal (end of clone). The KL amino acid sequence thereforehas the features of a transmembrane protein: an N-terminal signalpeptide, an extracellular domain, a transmembrane domain, and aC-terminal intracellular segment.

[0200] RNA blot analysis was performed to identify KL-specific RNAtranscripts in BALB/c 3T3 cells (FIG. 12). A major transcript of 6.5 kband two minor transcripts of 4.6 and 3.5 kb were identified on a blotcontaining poly(A)⁺ RNA by using the 1.4 kb KL cDNA as a probe.Identical transcripts were detected by using an end-labeledoligonucleotide derived from the N-terminal protein sequence. Thisresult then indicates that KL is encoded by a large mRNA that isabundantly expressed in BALB/c 3T3 cells.

[0201] The Soluble form of KL is a Ligand of the c-kit Receptor

[0202] The fibroblast-derived hematopoietic growth factor KL had beenshown to facilitate the proliferation of primary bone marrow mast cellsand peritoneal mast cells and to display erythroid burst-promotingactivity. To determine if KL is the ligand of the c-kit receptor, it wasfirst thought to demonstrate specific binding of KL to cells thatexpress high levels of the c-kit protein: mast cells (BMMC) and NIH ψ2cells expressing the c-kit cDNA. KL was labeled to high specificactivity with ¹²⁵I by using the modified chloramine T method (88).Analysis of the labeled material by SDS-PAGE showed a single band of28-30 kd (FIG. 13), and mast call proliferation assays indicated thatthe labeled material had retained its biological activity. Binding ofincreasing concentrations of ¹²⁵I-KL to NIH ψ2 cells expressing thec-kit cDNA, NIH ψ2 control cells, normal BMMC, and W/W, W/+, andW^(v)/W^(v) BMMC at 4° C. was measured. The results shown in FIG. 14indicate binding of labeled KL to NIH ψ2 c-kit cells and to +/+, W/+,and W^(v)/W^(v) mast cells, but not to NIH ψ2 control cells or W/W mastcells. The W^(v) mutation is the result of a missense mutation in thekinase domain of c-kit that impairs the in vitro kinase activity butdoes not affect the expression of the c-kit protein on the cell surface(36). By contrast, W results from a deletion due to a splicing defectthat removes the transmembrane domain of the c-kit protein; the proteintherefore is not expressed on the cell surface (36). Furthermore,binding of ¹²⁵I-KL could be completed with unlabeled KL and with twodifferent anti-c-kit antisera. These results indicated binding of¹²⁵I-labeled KL cells that express c-kit on their cell surface.

[0203] To obtain more direct evidence that KL is the ligand of the c-kitreceptor, it was determined if receptor-ligand complexes could bepurified by immunoprecipitation with c-kit antisera. This experimentrequires that a KL-c-kit complex be stable and not be affected by thedetergents used for the solubilization of the c-kit receptor. Precedentfor such properties of receptor-ligand complexes derives from theclosely related macrophage colony-stimulating factor (CSF-1) receptorand PDGF receptor systems (89). ¹²⁵I-KL was bound to receptors on BMMCby incubation at 4° C. Upon washing to remove free ¹²⁵I-KL, the cellswere solubilized by using the Triton X-100 lysis procedure andprecipitated with anti-v-kit and anti-c-kit rabbit sera conjugated toprotein A-Sepharose. 125I-KL was retained in immunoprecipitates obtainedby incubation with anti-kit sera but not with nonimmune controls, asshown by the analysis of the immune complexes by SDS-PAGE (FIG. 15A),where recovery of intact ¹²⁵I-KL was demonstrated from the samplescontaining the immune complexes prepared with anti-kit sera.

[0204] To further characterize the c-kit-KL receptor-ligand complexes,it was determined whether KL could be cross-linked to c-Kit. BMMC wereincubated with ¹²⁵I-KL, washed and treated with the cross-linkeddisucciminidyl substrate. Cell lysates were then immunoprecipitated withanti-v-kit antiserum and analyzed by SDS-PAGE. Autoradiography indicatedthree species: one at approximately 30 kd, representing KLcoprecipitated by not cross-linked to c-kit; one at 180-190 kd,corresponding to a covalently linked c-kit-KL monomer-monomer complex;and a high molecular weight structure that is at the interface betweenthe separating and stacking gels (FIG. 15B). Molecular structures ofsimilar size were observed if the cell lysates were separated directlyon SDS-PAGE without prior immunoprecipitation. Following precipitationwith nonimmune serum, no ¹²⁵I-labeled molecules were observed. Theformation of the high molecular weight structures was dependant on theincubation of KL with mast cells and was not observed by cross-linked KLwith itself. Taken together, these results provide evidence that KLspecifically binds to the c-kit receptor and is a ligand of c-kit.

[0205] Mapping of KL to the S1 Locus

[0206] To test whether KL is encoded at the S1 locus, recombinationanalysis was used to determine the map position of KL with respect to alocus that is tightly linked to S1. This locus is the site of thetransgene insertion in the transgenic line TG.EB (85). It was determinedthat genomic sequences cloned from the insertion site map 0.8±0.8 cMfrom S1. This therefore represents the closest known marker to S1.

[0207] To map KL with respect to the transgene insertion site,interspecific mapping analysis was employed utilizing crosses ofC57BL/6J mice with mice of the species Mus spretus. This strategyexploits the observation that restriction fragment length polymorphism(RFLPs) for cloned DNA are observed much more frequently between mice ofdifferent species than between different inbred laboratory strains (90).Linkage between the 1.4 kb KL cDNA probe and TIS Dra/SaI, a probe fromthe transgene insertion site, was assessed by scoring for concordance ofinheritance of their respective C57BL/6J or M. spretus alleles. Thesecould be easily distinguished by analyzing RFLPs that are revealed byTaq1 restriction digests. The results of this linkage analysis are shownin Table 2. Only one recombinant was found in 53 progeny. Thiscorresponds to a recombination percentage of 1.9±1.9. Since this valueis very close to the genetic distance measured between the transgeneinsertion site and S1, this result is consistent with the notion that KLmaps to the SS1 locus. TABLE 2 Mapping of the Position of the KL Gene byLinkage Analysis Using an Interspecific Cross Progeny ProbeNonrecominant Recombinant 1.4 kb KL cDNA B6 Sp B6 Sp TIS Dra/SaI B6 SpSp B6 32 20 0 1

[0208] The locus identified by KL was also examined in mice that carrythe original S1 mutation (50). For this purpose, the observation thatthe transgene insertion site locus is polymorphic in inbred strains wastaken advantage of, and was utilized to determine the genotype at S1during fetal development. C57BL/6J mice that carry the S1 mutationmaintained in the C3HeB/FeJ strain were generated by mating, and F1progeny carrying the S1 allele were intercrossed (C57BL/6JS1³CH/+S1^(C3)H/+). Homozygous SIISI progeny from this mating are anemicand are homozygous for a C3HeB/FeJ-derived RFLP at the transgeneintegration site (FIG. 16). Nonanemic mice are either heterozygous S1I+or wild type, and are heterozygous for the C3HeB/FeJ- andC57BL/6J-derived polymorphism or are homozygous for the C57BL/6Jpolymorphism, respectively. When genomic DNA from SII+ and SIISI micewas analyzed using the 1.4 kb KL cDNA probe, no hybridization to thehomozygous SIISI DNA was observed (FIG. 16). It thus appears that thelocus that encodes the KL protein is deleted in the S1 mutation. Thisfinding further supports the notion that KL is the product of the S1gene.

[0209] Experimental Discussion

[0210] The discovery of allelism between the c-kit proto-oncogene andthe murine W locus revealed the pleiotropic functions of the c-kitreceptor in development and in the adult animal. Furthermore, itprovided the first genetic system of a transmembrane tyrosine kinasereceptor in a mammal. Mutations at the S1 locus and at the c-kit/W locusaffect the same cellular targets. Because of the complementary andparallel properties of these mutations, it was proposed that the ligandof the c-kit receptor is encoded by the S1 locus.

[0211] The experiments reported herein provide evidence that the S1 geneencodes the ligand of the c-kit receptor. The evidence for thisconclusion is a follows. Based on the knowledge of the function of thec-kit receptor designated KL, a putative ligand of the c-kit receptordesignated KL was identified and purified (37). It was also demonstratedthat specific binding of KL to the c-kit receptor, as evidenced by thebinding of KL to cells expressing a functional c-kit receptor and theformation of a stable complex between KL and the c-kit protein.KL-specific cDNA clones were derived and it was shown that KL maps tothe S1 locus on mouse chromosome 10. In addition, it was alsodemonstrated that KL sequences are deleted in the genome of the S1mouse. Taken together, these results suggest that KL is encoded by theS1 locus and is the ligand of the c-kit receptor, thus providing amolecular basis for the S1 defect.

[0212] The amino acid sequence predicted from the nucleotide sequence ofthe KL cDNA clone suggests that KL is synthesized as an integraltransmembrane protein. The structural features of the primarytranslation product of KL therefore are akin to those of CSF-1. CSF-1 issynthesized as a transmembrane molecule, which is processed byproteolytic cleavage to form a soluble product that is secreted (91,44). Presumable, like CSF-1, KL is also synthesized as a cell surfacemolecule that may be processed to form a soluble protein. The proteinpurified from conditioned medium of BALB/c 3T3 cells then wouldrepresent the soluble form of KL that was released from the cellmembrane form by proteolytic cleavage. Although the post-translationalprocessing and expression of the KL protein have not yet beencharacterized, a cell surface-bound form of KL may mediate the cell-cellinteractions proposed for the proliferative and migratory functions ofthe c-kit/W receptor system. In agreement with the notion of a cellmembrane-associated form of KL, a soluble c-kit receptor-alkalinephosphatase fusion protein has been shown to bind to the cell surface ofBALB/c 3T3 cells but not to fibroblasts derived from SII/SI mice (14).

[0213] A most significant aspect of the identification of the ligand ofthe c-kit receptor lies in the fact that it will facilitate theinvestigation of the pleiotropic functions of c-kit. In thehematopoietic system c-kit/W mutations affect the erythroid and mastcell lineages, and an effect on the stem cell compartment has beeninferred as well. In erythroid cell maturation c-kit/KL plays anessential role, and this is best seen by the anemia of mutant animals.Furthermore, the number of CFU-E in fetal livers from W/W and SIISI^(d)animals is repressed, whereas the number of BFU-E remains normal,suggesting that c-kit/KL facilitates the progression from the BFU-E tothe CFU-E stage of differentiation (90, 35). In this regard, KL has beenshown to stimulate the proliferation and differentiation of BFU-E (day7) as well as earlier erythroid multipotential precursors in bonemarrow, which appear at later times in culture (day 14-20) (37).

[0214] An essential role for c-kit/KL in the proliferation,differentiation, and/or survival of mast cells in vivo has been inferredbecause of the absence of mast cells in W and S1 mutant mice (72, 73).The precise stage(s) at which c-kit/KL function is required in mast celldifferentiation is not known. The in vitro derivation of BMMC from bonemarrow or fetal liver does not require c-kit/KL function since BMMC canbe generated with comparable efficiency from both normal and W mutantmice (60). Applicants' demonstration of proliferation of BMMC andconnective tissue-type mast cells in response to KL indicates a role forc-kit/KL at multiple stages in mast cell proliferation anddifferentiation independent of IL-3 and IL-4, which are thought to bemediators of allergic and inflammatory responses (66). In the stem cellcompartment the affected populations possibly include the spleencolony-forming units (CFU-S), which produce myeloid colonies in thespleen of lethally irradiated mice, as well as cells with long-termrepopulation potential for the various cell lineages (80, 81, 82, 83).It will now be of interest to determine the effect of KL on theself-renewal or the differentiation potential of hematopoietic stem cellpopulations in vitro, possibly in combination with other hematopoieticgrowth factors, in order to identify the stage(s) where c-kit/KLfunctions in stem cells. Another possible function for c-kit might be tofacilitate the transition from noncycling to cycling cells (31). Theincreased radiation sensitivity of SIISI^(d) and of W/W^(v) mice mightsuggest such a role in stem cell dynamics; furthermore, the related PDGFreceptor is known to promote entry into the cell cycle.

[0215] In gametogenesis the W and S1 mutations affect the proliferationand the survival of primordial germ cells, and their migration from theyolk sac splanchnopleure to the genital ridges during early development.In postnatal gametogenesis c-kit expression has been detected inimmature and mature oocytes and in spermatogonia A and B as well as ininterstitial tissue (39). In melanogenesis c-kit/KL presumable functionsin the proliferation and migration of melanoblast from the neural crestto the periphery in early development as well as in mature melanocytes.The availability of KL may now facilitate in vitro studies of thefunction of the c-kit receptor in these cell systems. Themicroenvironment in which c-kit-expressing cells function is defectivein S1 mutant mice and is the presumed site where the c-kit ligand isproduced. Because of the extrinsic nature of the mutation, the preciseidentity of the cell types that produce KL in vivo is not known. Invitro systems that reproduce the genetic defect of the W and the S1mutations, however, have shed some light on this question. In thelong-term bone marrow culture system, SIISI^(d) adherent cells aredefective but the nonadherent hematopoietic cells are not, and in themast cell-fibroblast coculture system, SIISI^(d) fibroblasts aredefective but the mast cells are not (12, 16). The results from these invitro systems then would suggest that hematopoietic stromal cells andembryonic and connective tissue fibroblasts produce KL. The BALB/c 3T3cell line, which is of embryonic origin, expresses significant levels ofKL and was the source for its purification. Knowledge of KL-expressingcell types may help to evaluate if there is a function for c-kit in thedigestive tract, the nervous system, the placenta, and certaincraniofacial structures, sites where c-kit expression has beendocumented (35, 39). No SI or W phenotypes are known to be associatedwith these cell systems.

[0216] Interspecific backcrosses were used to establish close linkagebetween the KL gene, the S1 locus, and the transgene insertion locusTg.EB on mouse chromosome 10. A similar approach had previously beenused to map the Tg.EB locus in the vicinity of S1. The finding that theKL coding sequences are deleted in the original S1 allele, however,supports the identity of the S1 locus with the KL gene. The size of thedeletion in the S1 allele at this time is not known. It will beimportant to determine whether it affects neighboring genes as well.

[0217] The lack of KL coding sequences in the S1 allele indicates thatthis allele is a KL null mutation. When homozygous for the S1 allele,most mice die perinatally of macrocytic anemia, and rare survivors lackcoat pigmentation and are devoid of germ cells (5). This phenotypeclosely parallels that of severe c-kit/W loss-of-function mutations, inagreement with the ligand-receptor relationship of KL and c-kit.Although differences exist between SIISI and W/W homozygotes, e.g., ingerm cell development, S1 may have a more pronounced effect, and inhematopoiesis S1 may cause a more severe anemia; however, it is notknown if these differences are a result of different strain backgroundsor are possibly effects of the S1 deletion on neighboring genes (5).

[0218] The original W mutation is an example of a c-kit null mutation(36). When heterozygous with the normal allele, WI+ mice typically havea ventral spot but no coat dilution and no effects on hematopoiesis andgametogenesis. The weak heterozygous phenotype of WI⁺ mice is incontrast to the phenotype of heterozygous SII⁺ mice, which have moderatemacrocytic anemia and a diluted coat pigment in addition to a ventralspot and gonads that are reduced in size. Thus 50% gene dosage of KL islimiting and is not sufficient for normal function of the c-kitreceptor, yet 50% dosage of the c-kit receptor does not appear to belimiting in most situations.

[0219] The c-kit receptor system functions in immature progenitor cellpopulations as well as in more mature cell types in hematopoiesis,gametogenesis, and melanogenesis. Severe S1 or W mutations may block thedevelopment of these cell lineages, and therefore a function for thec-kit receptor in more mature cell populations would not be evident. S1and W mutations in which c-kit/KL function is only partially impairedoften reveal effects in more mature cell populations. Numerous weak S1alleles are known. Their phenotypes, e.g., in gametogenesis andmelanogenesis, will be of great value in the elucidation of thepleiotropic functions of the c-kit receptor system.

[0220] EXPERIMENT NUMBER 3—KL-1 AND KL-2

[0221] Experimental Materials

[0222] Mice and Tissue Culture

[0223] WBB6+/+, C57BL/6J and 129/Sv-S1^(d)/+ mice were obtained from theJackson Laboratory (Bar Harbor, Me.) (52). 129/Sv-S1^(d)/+ male andfemale mice were mated and day 14 fetuses were obtained and used for thederivation of embryonic fibroblasts according to the method of Todaroand Green (54). Mast cells were grown from bone marrow of adult +/+ micein RPMI-1640 medium supplemented with 10% fetal calf serum (FCS),conditioned medium from WEHI-3B cells, non-essential amino acids, sodiumpyruvate, and 2-mercapto-ethanol (RPMI-Complete (C)) (36). Balb/3T3cells (1) were grown in Dulbecco's Modified MEM (DME) supplemented with10% calf serum (CS), penicillin and streptomycin. COS-1 cells (18) wereobtained from Dr. Jerrard Hurwitz (SKI) and were grown in DMEsupplemented with 10% fetal bovine serum, glutamine, penicillin andstreptomycin.

[0224] Production of anti-KL Antibodies

[0225] Murine KL was purified from conditioned medium of Balb3T3 cellsby using a mast cell proliferation assay as described elsewhere (37). Inorder to obtain anti-KL antibodies one rabbit was immunizedsubcutaneously with 1 μg of KL in complete Freund's adjuvant. Threeweeks later the rabbit was boosted intradermally with 1 μg in incompleteFreunds adjuvant. Serum was collected one week later and then biweeklythereafter. The ¹²⁵I-labelled KL used for this purpose was iodinatedwith chloramine T with modifications of the method of Stanley andGilbert as described previously (38).

[0226] cDNA Library Screening

[0227] Poly(A) RNA was prepared by oligo(dT)-cellulose chromatographyfrom total RNA of Balb/c 3T3 fibroblast. A custom made plasmid cDNAlibrary was then prepared by Invitrogen Inc. Essentially,double-stranded cDNA was synthesized by oligo dT and random priming.Non-palindromic BstXI linkers were ligated to blunt-ended cDNA and upondigestion with BstXI the cDNA was subcloned into the expression plasmidpcDNAI (Invitrogen). The ligation reaction mixture then was used totransform E. coli MC1061/P3 by the electroporation method to generatethe plasmid library. The initial size of the library was approximately10⁷ independent colonies. For screening of the plasmid library anend-labelled oligonucleotide probe described previously was used (38).Hybridization was done in 6×SSC at 63° C. and the final wash of thefilters was in 2× SSC and 0.2% SDS at 63° C. The inserts of recombinantplasmids were released by digestion with HindII and XbaI and thensubcloned into the phage M13 mp18 for sequence analysis.

[0228] PCR Amplification (RT-PCR) and Sequence Determination

[0229] Total RNA from tissues and cell lines was prepared by theguanidium isothiocyanate/CsCl centrifugation method of Chirgwin (10).The RT-PCR amplification was carried out essentially as describedpreviously (38). The following primers were used for RT-PCR: Primer #1:5′-GCCCAAGCTTCGGTGCCTTTCCTTATG-3′ (nt. 94- 107); Primer #2:5′-AGTATCTCTAGAATTTTACACCTCTTGAAATTCTCT-3′ (nt. 907- 929); Primer #3:5′-CATTTATCTAGAAAACATGAACTGTTACCAGCC-3′ (nt. 963- 978); Primer #4:5′-ACCCTCGAGGCTGAAATCTACTTG-3′ (nt. 1317- 1333).

[0230] For cDNA synthesis, 10 μg of total RNA from cell lines or tissuesin 50 μl of 0.05 mM Tris-HCl (pH 8.3), 0.75 M KCl, 3 mM MgCl₂, 10mM DTT,200 μM dNTP's and 25 U of RNAsin (BRL) was incubated with 50 pmole ofantisense primer and 400 U of Moloney murine leukemia virus reversetranscriptase (BRL) at 37° C. for 1 hour. The cDNA was precipitated byadding {fraction (1/10)} volume of 3 M NaOAc (pH 7.0) and 2.5 volume ofabsolute ethanol and resuspended in 50 μl of ddH₂O. PCR was carried outfor 30 cycles in 100 μl of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mMMgCl₂, 0.01% (w/v) gelatin, 200 μM dNTP's, 500 pmole of both sense andantisense primers and 2.5 U of Taq polymerase (Perkin-Elmer-Cetus).HindII sites and XbaI sites were placed within the sense—and antisenseprimers respectively. The amplified DNA fragments were purified byagarose gel electrophoresis, digested with the appropriate restrictionenzymes, and subcloned into M13mp18and M13mp19for sequence analysis(49). The KL-1, KL-2, KL-S and KL-S1^(d) PCR products were digested withHindIII and XbaI and subcloned into the expression plasmids pCDM8 orpcDNAI (Invitrogen). Miniprep plasmid DNA was prepared by thealkaline-lysis method (48) followed by phenol-chloroform extraction andethanol precipitation. Maxiprep plasmid DNA used for the transfection ofCOS-1 cells was prepared by using the “Qiagen” chromatography columnprocedure.

[0231] RNase Protection Assay

[0232] A riboprobe for RNAse protection assays was prepared bylinearizing the KL-1 containing pcDNAI plasmid with SpeI. The antisenseriboprobe was then synthesized by using SP6 polymerase according to thePromega Gemini kit. Riboprobe labelled to high specific activity wasthen hybridized to 10 or 20 μg of total RNA in the presence of 80%formamide at 45° C. overnight. The hybridization mixture was digestedwith RNAse A and T1 (Boehringer-Mannheim) and treated with proteinase K(48) and the protected labelled RNA fragments were analyzed on a 4%urea/polyacrylamide gel. Autoradiograms of RNAse protection assay wereanalyzed by densitometry and parts of the films were reconstructed on aPhosphoImage analyzer (Molecular Dynamics) for better resolution.

[0233] Transient Expression of “KL” cDNAs in COS-1 Cells

[0234] For transient expression of KL cDNAs COS-1 cells were transfectedwith the DEAE-dextran method described previously (20) with minormodifications. Briefly, COS-1 cells were grown to subconfluence one daybefore use and were trypsinized and reseeded on 150 mm petri dishes at adensity of 6×10⁶ cells per dish. After 24 hours, the cells had reachedabout 70% confluence and were transfected with 5 μg of plasmid DNA inthe presence of 10% DEAE-dextran (Sigma) for 6 to 12 hours. Mediumcontaining plasmid DNA was removed and the cells were chemically shockedwith 10% DMSO/PBS⁺⁺ for exactly 1 minute. Residual DMSO was removed bywashing the cells with pBS⁺⁺ twice. Transfected COS-1 cells were grownin DME plus 10% fetal calf serum, 100 mg/ml L-glutamine, andantibiotics.

[0235] Pulse Chase and Immunoprecipitation Analysis of “KL”Proteins

[0236] Transfected COS-1 cells were used for pulse-chase experiments 72hours after the transfection. Cells were incubated with methionine-freeDME containing 10% dialyzed fetal calf serum for 30 minutes and labelledwith ³⁵S-methionine (NEN) at 0.5 mCi/ml. At the end of the labellingperiod, the labelling medium was replaced with regular medium containingan excess amount of methionine. In order to determine the effect ofphorbol 12-myristate 13-acetate (PMA) and A23187 on the proteolyticcleavage of KL, 1 μM PMA or 1 μM A23187 was added to the transfectedcells at the end of the labelling period after replacement of thelabelling medium with regular medium. The cells and supernatants werecollected individually at the indicated times for immunoprecipitationanalysis. Cell lysates were prepared as described previously (35) in 1%Triton -100, 20 mM Tris (pH 7.5), 150 mM NaCl, 20 mM EDTA, 10% glyceroland protease inhibitors phenylmethyl sulfonyl chloride (1 mM) andleupeptin (20 μg/ml). For the immunoprecipitation analysis of KL proteinproducts the anti-mouse KL rabbit antiserum was used. The anti-KL serumwas conjugated to protein-A Sepharose (Pharmacia) and washed 3 timeswith Wash A (0.1% Triton X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, 10%glycerol). Anti-KL serum-protein A sepharose conjugate was incubatedwith supernatant and cell lysate at 4° C. for at least 2 hours. Theimmunoprecipitates then were washed once in Wash B (50 mM Tris, 500 mMNaCl, 5 mM EDTA, 0.2% Triton X-100), 3 times in Wash C (50 mM Tris, 500mM NaCl, 0.1% Triton X-100, 0.1% SDS, 5 mM EDTA) and once in Wash D (10mM Tris, 0.1% Triton X-100). For gel analysis immunoprecipitates weresolubilized in SDS sample buffer by boiling for 5 minutes, and analyzedby SDS-PAGE (12%) and autoradiography.

[0237] Determination of Biological Activity of Soluble KL

[0238] Mast cells were grown from bone marrow of adult WBB6 +/+ mice inRPMI-1640 medium supplemented with 10% fetal calf serum, conditionedmedium from WEHI-3B cells, non-essential amino acids, sodium pyruvateand 2-mercaptoethanol (RPMI-Complete) as described previously (37).Non-adherent cells were harvested by centrifugation and refed weekly andmaintained at a cell density of <7×10⁵ cells/ml. The mast cell contentof cultures was determined weekly by staining cytospin preparations with1% toluidine blue in methanol. After 4 weeks, cultures routinelycontained >95% mast cells and were used for proliferation assay.Supernatants from transfected COS-1 cells were collected from 48 to 72hours after transfection. The biological activity of soluble KL in thesupernatants was assessed by culturing BMMCs with different dilutions ofCOS-1 cell supernatants in the absence of IL-3. BMMCs were washed threetimes with complete RPMI and grown in 0.2% IL-3. The following day,cells were harvested and suspended in complete RPMI (minus IL-3) and 10⁴BMMCs in 100 μl/well were seeded in a 96-well plate. Equal volume ofdiluted supernatant was added to each well and cultures were incubatedfor 24 hours at 37° C., 2.5 μCi of [³H]-thymidine/well was then addedand incubation was continued for another 6 hours. Cells were harvestedon glass fiber filters (GF/C Whatman) and thymidine incorporation wasdetermined in a scintillation counter. Assays were performed intriplicate and the mean value is shown. Standard deviations ofmeasurements typically did not exceed 10% of the mean values.

[0239] Experimental Results

[0240] Alternatively Spliced Transcript of KL Encodes a TruncatedTransmembrane form of the KL Protein

[0241] A cDNA clone, which had been isolated from a mouse 3T3 fibroblastlibrary and contained most of the KL coding sequences (267 amino acids),has been described herein. In an attempt to obtain the complete cDNAsequences corresponding to the 6.5 kb KL mRNA, a plasmid cDNA librarywas constructed by using polyA⁺ RNA from Balb/c3T3 fibroblasts. Theplasmid vector pcDNAI which was used for this purpose is a mammalianexpression vector in which cDNA inserts are expressed from a CMVpromoter and contains an SV40 origin of replication for transientexpression in COS cells (Invitrogen). The library was screened witholigonucleotide probes corresponding to N-terminal and C-terminal KLcoding sequences as described herein. A cDNA clone which contains thecomplete KL coding sequences as well as 5′ and 3′ untranslated sequenceswas obtained. The nucleotide sequence of this clone (FIG. 17) is inagreement with the previously published sequences except for a singlebase change at position 664 which results in the substitution of serine206 to alanine (2,38).

[0242] The analysis of murine KL cDNA clones by Anderson andcollaborators indicated a spliced cDNA with an inframe deletion of 48nucleotides suggesting the presence of alternatively spliced KL RNAtranscripts in KL expressing cells (2). To identify alternativelyspliced KL RNA transcripts in RNA from tissues and cell lines, theRT-PCR method was used. The primers used corresponded to the 5′ and 3′untranslated regions of the KL cDNA and were modified to contain uniquerestriction sites. Electrophoretic analysis of the RT-PCT reactionproducts shown in FIG. 18 indicates a single fragment of approximately870 bp in the samples from Balb3T3 cells and brain, whereas in thesamples from spleen, testis and lung two fragments were seen,approximately 870 and 750 bp in size. For further analysis the two PCRreaction products were subcloned into the mammalian expression vectorpCDM8. DNA sequence analysis first indicated that the larger PCR productcorresponds to the known KL cDNA sequence, subsequently referred to asKL-1. In the smaller PCR product, however, a segment of 84 nucleotidesof the KL coding sequences was lacking, generating an inframe deletion.The deletion endpoints corresponded to exon boundaries in the rat andthe human KL genes and it is quite likely that these boundaries are alsoconserved in the mouse gene (27). Therefore, the smaller PCR productappeared to correspond to an alternatively spliced KL RNA transcript,designated KL-2. The exon missing in KL-2 precedes the transmembranedomain; it contains one of the four N-linked glycosylation sites andincludes the known C-terminus (Ala-166 and Ala-167) of the soluble formof KL (58). KL-2 therefore is predicted to encode a truncated version ofKL-1 which is presumably synthesized as a transmembrane protein (FIGS.17 and 19).

[0243] KL-2 Is Expressed In A Tissue Specific Manner

[0244] The alternatively spliced transcript KL-2 had been detected inspleen, testis and lung RNA, but not in fibroblasts and brain RNA,suggesting that the expression of KL-2 may be controlled in a tissuespecific manner. In order to address this question in more detail thesteady state levels of KL-1 and KL-2 RNA transcripts in RNA weredetermined from a wide variety of tissues by using an RNAse protectionassay. pcDNAI plasmid containing the KL-1 cDNA was linearized with SpeIin order to generate an RNA hybridization probe of 625 nucleotides byusing SP6 RNA polymerase. The probe was hybridized with 20 μg of totalRNA from Balb/c 3T3 fibroblasts, brain, spleen and testis of a 40 daysold mouse, as well as from brain, bone marrow, cerebellum, heart, lung,liver, spleen and kidney of an adult mouse and placenta (14 days p.c.).The samples then were digested with RNAse and the reaction productsanalyzed by electrophoresis in a 4% urea/polyacrylamide gel. In theseexperiments KL-1 mRNA protected a single fragment of 575 bases, whileKL-2 mRNA protected fragments of 449 and 42 nucleotides. As shown inFIG. 20, in Balb/c3T3 fibroblasts, KL-1 is the predominant transcriptwhereas the KL-2 is barely detectable. In brain and thymus KL-1 is thepredominant transcript, but in spleen, testis, placenta, heart andcerebellum both KL-1 and KL-2 transcripts are seen in variable ratios.The ratio of the KL-1 to KL-2 in tissues determined by densitometry inbrain is 26:1, in bone marrow 3:1, in spleen 1.5:1 and in testis (40days p.n.) 1:2.6. These results suggest that the expression of KL-1 andKL-2 is regulated in a tissue-specific manner.

[0245] Biosynthetic Characteristics of KL Protein Products in COS Cells

[0246] Although KL was purified from conditioned medium of Balb/c 3T3cells and is a soluble protein, the predicted amino acid sequences forKL-1 and KL-2 suggest that these proteins are membrane-associated. Inorder to investigate the relationship of KL-S with the KL-1 and KL-2protein products their biosynthetic characteristics were determined. TheKL-1 and KL-2 cDNAs, prepared by RT-PCR, were subcloned into the HindIIIand XbaI sites of the expression vectors pcDNAI or pCDM8 for transientexpression in COS-1 cells. To facilitate transient expression of theKL-1 and KL-2 protein products COS-1 cells were transfected with theKL-1 and KL-2 plasmids by using the DEAE-dextran/DMSO protocol asdescribed herein. KL protein synthesis in the COS-1 cells was shown tobe maximal between 72 to 96 hours subsequent to the transfection. Inorder to determine the biosynthetic characteristics of the KL-1 and KL-2proteins pulse-chase experiments were carried out. 72 hours subsequentto transfection, cultures were labeled with ³⁵S-methionine (0.5mCi/ml)for 30 minutes and then chased with regular medium. The cell lysate andsupernatants then were collected at the indicated times and processedfor immunoprecipitation with anti-KL antiserum, prepared by immunizingrabbits with purified murine KL, and analysis by SDS-PAGE (12%). Incells transfected with the KL-1 plasmid, at the end of the labellingperiod, KL specific protein products of 24, 35, 40 and 45 kD are found(FIG. 21). These proteins presumably represent the primary translationproduct and processed KL protein products which are progressivelymodified by glycosylation. Increasingly longer chase times reveal the 45kD form as the mature KL protein product and it is quite likely thatthis protein represents the cell membrane form of KL. In the supernatantbeginning at 30 minutes a 28 kD KL protein product is seen which, withincreasing time, increases in amount. Two minor products of 38 and 24 kDwere also found with increasing time. These results are consistent withthe notion that KL-1 is first synthesized as a membrane-bound proteinand then released into the medium probably through proteolytic cleavage.

[0247] A pulse-chase experiment of COS-1 cells transfected with the KL-2plasmid is shown in FIG. 20. The KL-2 protein products are processedefficiently to produce products of 32 kD and 28 kD which likely includethe presumed cell membrane form of KL-2. The cell membrane form of KL-2is more stable than the corresponding KL-1 protein with a half-life ofmore than 5 hours. In the cell supernatant, after 3 hours, a solubleform of KL-2 of approximately 20 kD is seen. The appearance andaccumulation of the soluble form of KL-2 in the cell supernatant isdelayed compared with that of KL-1 in agreement with less efficientproteolytic processing of the KL-2 protein product. In KL-2, as a resultof alternative splicing, sequences which include the known C-terminus ofthe soluble form of KL and thus the presumed cleavage site of KL-1 ismissing. Proteolytic cleavage of KL-2, therefore, presumably involves asecondary cleavage site which is present in both KL-1 and KL-2, eitheron the N-terminal or C-terminal side of the sequences encoded by thedeleted exon. A 38 kD KL-1 protein product seen in the supernatant mayrepresent a cleavage product which involves a cleavage site near thetransmembrane domain (FIG. 19).

[0248] Proteolytic Processing of KL-1 And KL-2 in COS Cells is Modulatedby PMA and the Calcium Ionophore A23187

[0249] The protein kinase C inducer PMA is known to facilitateproteolytic cleavage of cell membrane proteins to produce soluble formsof the extra-cellular domain of these proteins as shown with theexamples of the CSF-1 receptor, the c-kit receptor and TGF-α (13,4). Theeffect of PMA treatment on the biosynthetic characteristics of KL-1 andKL-2 in COS-1 cells has been determined. The pulse-chase experimentsshown in FIG. 22B indicate that PMA induces the rapid cleavage of bothKL-1 and KL-2 with similar kinetics and that the released KL-1 and KL-2protein products are indistinguishable from those obtained in theabsence of inducer. These results suggest that the proteolytic cleavagemachinery for both KL-1 and KL-2 is activated similarly be PMA. On onehand this may mean that two distinct proteases, specific for KL-1 andKL-2 respectively, are activated by PMA or alternatively, that there isone protease which is activated to a very high level which cleaves bothKL-1 and KL-2 but with different rates. The major cleavage site in KL-1based on the known C-terminal amino acid sequence of rat KL, includesamino acids PPVA A SSL (186-193) and may involve an elastase like enzyme(22,34). The recognition sequence in KL-2, based on the argumentspresented above, presumably lies C-terminal of the deleted exon andtherefore might include amino acids RKAAKA (202-207) and thus couldinvolve an enzyme with a specificity similar to the KL-1 protease,alternatively, it could be a trypsin-like protease. The effect of thecalcium ionophore A23187 on KL cleavage has been determined. Both KL-1and KL-2 cleavage is accelerated by this reagent indicating thatmechanisms that do not involve the activation of protein kinase C canmediate proteolytic cleavage of both KL-1 and KL-2 (FIG. 22 C).

[0250] Biological Activity of the Released KL Protein Products

[0251] To test the biological activity of the released KL proteinproducts, the supernatants of transfected COS-1 cells were collected 72hours after transfection and assayed for activity in the mast cellproliferation assay. Bone marrow derived mast cells (BMMC) wereincubated for 24 hours with different dilutions of the collectedsupernatants and assayed for ³H-thymidine incorporation as describedpreviously (FIG. 23). Supernatants from KL-l transfectants produced 3 to5 times more activity than KL-2 transfectants in agreement with thedifferential release of soluble KL from KL-1 and KL-2. Importantly theproteins released from both the KL-1 and the KL-2 transfectants appearedto display similar specific activities in the mast cell proliferationassay.

[0252] The Steel Dickie Allele Results from a Deletion of C-terminal KLCoding Sequences Including the Transmembrane and the Cytoplasmic Domains

[0253] Mice homozygous for the S1^(d) allele are viable, in contrast tomice homozygous for the S1 allele, although they lack coat pigment, aresterile and have macrocytic anemia. The c-kit receptor system in thesemice, therefore, appears to display some residual activity. The S1^(d)mutation affects the three cell lineages to similar degrees suggestingthat the mutation affects an intrinsic property of KL. Thus, toinvestigate the molecular basis of S1^(d), the KL coding sequences werefirst characterized in this allele by using PCR cloning technology.Primary embryo fibroblasts from an S1^(d)/+ embryo were derived bystandard procedures. RNA prepared from S1^(d)/+ embryo fibroblasts anddifferent primers then were used to amplify the S1^(d) KL coding regionpaying attention to the possibility that S1^(d) is a deletion mutation.RT-PCR amplification by using S1^(d)/+ total RNA and primers 1 and 2produced one DNA fragment that migrated with a mobility identical tothat of the product obtained from +/+ fibroblast RNA and sequencedetermination showed it to be indistinguishable from the known KLsequence. This fragment therefore presumably represented the normalallele. When primers 1 and 3 or 1 and 4 were used a faster migrating DNAfragment was amplified was well (FIG. 18). Both the 850 and 1070 bp DNAfragments obtained with primers 1+3 and 1+4 were subcloned into pCDM8and then sequenced. In the KL-S1^(d) cDNA the segment from nucleotides660 to 902 of the wild-type sequence is deleted, instead, a sequence of67 bp was found to be inserted (FIG. 17). The deletion insertion resultsin a termination codon three amino acids from the 5′ deletion endpoint.The predicted amino acid sequence of KL-S1^(d) cDNA consists of aminoacids 1-205 of the known KL sequence plus 3 additional amino acids(FIGS. 17 and 19). The KL-S1^(d) amino acid sequence includes all fourN-linked glycosylation sites and all sequences contained in the solubleform of KL, while the transmembrane and the cytoplasmic domains ofwild-type KL-1 are deleted. Consequently, the KL-S1^(d) protein productis a secreted protein, which displays biological activity.

[0254] Biosynthetic Characteristics And Biological Activity Of TheKL-S1^(d) and KL-S Protein Products

[0255] For comparison with the KL-S1^(d) protein product, a truncatedversion of KL-1 was made, designated KL-S, in which a termination codonwas inserted at amino acid position 191 which is the presumed C-terminusof the soluble KL protein. COS-1 cells were transfected with theKL-S1^(d) and the KL-S plasmids and pulse-chase experiments were carriedout to determine the biosynthetic characteristics of the two proteinproducts. The KL-S1^(d) protein product is rapidly processed, presumablyby glycosylation and then secreted into the medium, where the major 30kD species is found after as early as 30 minutes of chase time and thenincreases in amount thereafter (FIG. 24). The biosyntheticcharacteristics of the KL-S protein products are very similar to thoseof KL-S1^(d) (FIG. 24). Again, with increasing time increasing amountsof secreted material are detected in the medium, conversely the cellassociated KL-S protein products decrease with time. To assess thebiological activity of the secreted KL-S1^(d) and KL-S protein products,mast cell proliferation assays were performed. The medium fromtransfected COS-1 cells was collected 72 hours after transfection andthen different dilutions were used to assess proliferative potentialconferred on BMMC in the absence of IL-3. Both samples containedsignificant biological activity that exceeded that of KL-1 to somedegree (FIG. 23). Taken together, these results demonstrateconvincingly, that the KL-S1^(d) protein products are secreted and arebiologically active.

[0256] Experimental Discussion

[0257] The demonstration of allelism between c-kit and the murine Wlocus brought to light the pleiotropic functions of the c-kit receptorin development and in the adult animal and facilitated theidentification of its ligand KL. The recent discovery of allelismbetween KL and the murine steel locus, furthermore provided a molecularnotion of the relationship between the W and the S1 mutations which hadbeen anticipated by mouse geneticists based on the parallel andcomplementary phenotypes of these mutations. The predicted transmembranestructure of KL implicated that, both, membrane-associated and solubleforms of KL play significant roles in c-kit function. In thisapplication, experimental evidence for this conjecture is provided.

[0258] First, it is shown that the soluble form of KL is generated byefficient proteolytic cleavage from a transmembrane precursor, KL-1.Second, an alternatively spliced version of KL-1, KL-2, in which themajor proteolytic cleavage site is removed by splicing, is shown toproduce a soluble biologically active form of KL as well, although, withsomewhat diminished efficiency. Third, cleavage of KL-1 and KL-2 inCOS-1 cells is a process that can be modulated. Fourth, KL-1 and KL-2are expressed in a tissue-specific manner. Furthermore, the viableS1^(d) mutation was shown to be the result of a deletion that includesthe C-terminus of the KL coding sequence including the transmembranedomain generating a biologically active secreted form of KL. Thephenotype of mice carrying the S1^(d) allele provides further supportfor the concept for a role for both the secreted and the cell membrane-associated forms of KL in c-kit function.

[0259] Because of the close evolutionary relationship of c-kit withCSF-1R it was reasonable to predict a relationship between thecorresponding growth factors, KL and CSF-1, in regards to bothstructural and topological aspects. Alternatively spliced forms of CSF-1mRNAs are known to encode protein products which differ in sequencesN-terminal of the transmembrane domain, a spacer segment of 298 aminoacids located in between the ligand portion and the transmembrane domainof the protein (43). In addition, alternatively spliced CSF-I RNAtranscripts differ in their 3′ untranslated regions (21). Analysis of KLRNA transcripts in several tissues identified an alternatively splicedKL RNA in which, similar to the situation in CSF-1, the spacer betweenthe presumed ligand portion and the transmembrane domain is deleted.Interestingly, the expression of this alternatively spliced RNA productis controlled in a tissue specific manner. A recent comparative analysisof the ligand portions of KL and CSF-1 indicates structural homologybetween the two proteins based on limited amino acid homology and thecomparison of corresponding exons and matching of “exon-encodedsecondary structure” (4). Furthermore, the super position of 4 a-helicaldomains and cysteine residues which form intra-molecular disulfide bondsimplies related tertiary structures for the ligand domains of KL andCSF-1; and the homology seen in the N-terminal signal peptides, thetransmembrane domains and the intracellular domains of the two proteinsmay indicate that these domains fulfill important related functions inthe two proteins. These results strengthen the notion of an evolutionaryrelationship and structural homology between KL and CSF-1.

[0260] A unique feature of KL is its predicted tripartite structure as atransmembrane protein. Both forms of KL, KL-1 and KL-2, are synthesizedas transmembrane proteins which are processed by proteolytic cleavage torelease a soluble biologically active form of KL; although, theprocessing step in the two forms follows differing kinetics, asdetermined in the COS cell system. Proteolytic cleavage of the KL-1protein is very efficient, in contrast, the KL-2 protein is more stableor resistant to proteolytic cleavage.

[0261] The sequences encoded by the deleted exon, amino acids 174-201include the C-terminus of the soluble KL protein and the presumedproteolytic cleavage site (27). A secondary or alternate proteolyticcleavage site is therefore presumably being used to generate the solubleKL-2 protein and this cleavage might involve another protease. Theinduction of proteolytic cleavage of KL-1 and KL-2 in COS-1 cells by theprotein kinase C activator PMA and by the calcium ionophore A23187suggests that in different cell types this process may be subject todifferential regulation. Interestingly, the soluble KL-2 proteindisplays normal biological activity indicating that the sequencesencoded by the deleted exon are not essential for this activity.

[0262] On one hand, KL-1 and KL-2 in their membrane associated versionsmay function to mediate their signal by cell-cell contact or,alternatively, they might function as cell adhesion molecules (19, 26).On the other hand, the soluble forms of KL are diffusible factors whichmay reach the target cell and its receptor over a relatively short orlonger distances. But the soluble forms of KL might also becomeassociated with, or sequestered in the extracellular matrix, in ananalogous fashion to FGF, LIF or int-1, and thus function over a shortdistance similar to the membrane-associated form (8,33,42). When cellmembrane-associated, KL may be able to provide or sustain highconcentrations of a localized signal for interaction withreceptor-carrying target cells. In turn the soluble form of KL mayprovide a signal at lower and variable concentrations. c-kit is thoughtto facilitate cell proliferation, cell migration, cell survival andpost-mitotic functions in various cell systems. By analogy with theCSF-1 receptor system, the cell survival function and cell migrationmight require lower concentrations of the factor than the cellproliferation function (55). The cell membrane-associated and thesoluble forms of KL then may serve different aspects of c-kit function.Both the CSF-1 receptor and c-kit can be down-regulated by proteinkinase C mediated proteolytic release of the respective extracellulardomains (13). The functional significance of this process is not knownbut it has been hypothesized that the released extracellular domain ofthese receptors may neutralize CSF-1 and KL, respectively, in order tomodulate these signals. In some ways proteolytic cleavage of KL resultsin a down modulation of c-kit function and the processes, therefore, maybe considered as complementary or analogous. In summary, the synthesisof variant cell membrane-associated KL molecules and their proteolyticcleavage to generate soluble forms of KL provide means to control andmodulate c-kit function in various cell types during development and inthe adult animal.

[0263] A unique opportunity to evaluate the role of the soluble form ofKL during development and in adult animals was provided through thecharacterization of the molecular basis of the S1^(d) mutation. TheS1^(d) allele encodes a secreted version of the KL protein and nomembrane associated forms as a result of a deletion which includes thetransmembrane domain and the C-terminus of KL. The biologicalcharacteristics of S1^(d)/S1^(d) and S1/S1^(d) mice, therefore shouldgive clues about the role of the soluble and the membrane-associatedforms of KL. S1/S1^(d) mice produce only the S1^(d) protein, since theS1 allele is a KL null-mutation (11,38).

[0264] These mice are viable and are characterized by a severemacrocytic anemia, lack of tissue mast cells, lack of coat pigmentationand infertility. In most aspects of their mutant phenotype, these miceresemble W/W^(v) mice (47,51). However some significant differencesexist. The anemia of S1/S1^(d) mice appear to be more sensitive tohypoxia than W/W^(v) mice (46, 47). In regards to gametogenesis inW/W^(v) mice primordial germ cells do not proliferate and theirmigration is retarded (32). In S1/S1^(d) embryos primordial germ cellssimilar to W/W^(v) embryos do not proliferate, however the remainingcells appear to migrate properly and they reach the gonadal ridges atthe appropriate time of development (29,51). From these experiments onemight hypothesize that the S1^(d) KL protein product is able to sustaincell migration but not cell proliferation and consequently the cellmembrane form of KL therefore may play a critical role in theproliferative response of c-kit. Furthermore, S1/S1^(d) fibroblasts donot support the proliferation and maintenance of bone marrow mast cellsin the absence of IL-3, in contrast to normal embryo fibroblasts whichhave this property (16). Provided that the S1/S1^(d) fibroblast indeedsynthesize the S1^(d) protein products, the inability of the S1/S1^(d)fibroblasts to support the proliferation of mast cells, on one hand, mayindicate that the amount of soluble KL-S1^(d) protein which is releasedby these cells is not sufficient to facilitate proliferation; on theother nand, these results may suggest that there is a critical role forthe cell membrane associated form of KL in this process.

[0265] KL In Combination With IL-1, IL-3, G-CSF, GM-CSF

[0266] We have used murine KL (recombinant murine c-kit ligand) innormal murine bone marrow cultures and observed very few myeloidcolonies stimulated with KL alone, but a substantial increase in bothcolony number and size was seen with combinations of KL and G-CSF,GM-CSF, and IL-3, but not with M-CSF (103). In HPP-CFC assays usingmarrow 24 hours post 5-FU treatment, increasing colony stimulation wasseen with combinations of cytokines. KL plus either G-CSF, GM-CSF, IL-3,IL-7, or IL-6 was effective and combinations of three or four factorswere even more effective in stimulating HPP-CFC, CSF's or IL-3 combinedwith IL-1, IL-6, and KL were maximally effective. FIG. 25 shows HPP-CFCstimulated by cytokine combinations in cultures of 4-day post 5-FUmurine marrow. In dual cytokine combinations, IL-1 plus GM-CSF or IL-3stimulated comparable numbers of HPP-CFC, as did KL plus IL-1 or KL plusIL-3, but three factor combinations of IL-1 plus KL and either G-CSF, orIL-3 were maximally effective. Delta or secondary CFU assay for earlyhematopoietic cells: Murine studies. The delta assay involves theshort-term (7-day) suspension culture of bone marrow depleted ofcommitted progenitors and enriched for early stem cells in the presenceof various cytokines to promote survival, recruitment, differentiation,and expansion of stem cells and progenitor cells is measured in asecondary clonogenic assay. 5-FU-resistant stem cells are assayed in aprimary HPP-CFC assay with multiple cytokine stimuli as well as inconventional CFU-GM assays with single CSF stimuli. After suspensionculture secondary HPP-CFC and CFU-GM assays are performed. Threeparameters are routinely measured. First is the amplification oflineage-restricted progenitors determined by the total CFU-GM responsiveto a single CSF species (eg, G-CSF) in the primary culture (input)divided into total number of secondary CFU-GM responsive to the same CSFspecies in the secondary culture (output). Second is the ratio ofHPP-CFC input divided into the total number of CFU-GM progenitors in thesecondary assay. Because CFU-GM are presumed to derive from earlierprecursors, i.e., HPP-CFC, this ratio gives the indication of stem cellto progenitor cell differentiation. Finally, the ratio of HPP-CFC inputdivided into the total number of secondary HPP-CFC is determined. Thisparameter is the best measure of stem cell self-renewal, particularly ifthe HPP-CFC stimulus in the primary and secondary cultures is acombination of IL-1, IL-3 and KL.

[0267] In earlier studies (before the availability of KL), varyingdegrees of expansion in the number of CFC-GM responsive to single CSFspecies, and in HPP-CFC-1 and 2, were seen when IL-1 was combined withM-CSF (20- to 30-fold increases), with G-CSF (50- to 100-foldincreases), with 200-fold increases) IL-3 and GM-CSF produced a limiteddegree of progenitor cell expansion whereas M-CSF and G-CSF did not.IL-6 was less effective than IL-1 in synergizing with M-CSF, GM-CSF, orG-CSF but was equally effective in synergizing with IL-3. IL-1 plus IL-6showed additive or supradditive interactions with the three CSF's andIL-3. When KL (prepared as described herein or alternatively prepared asdescribed in PCT International Publication No. WO 92/00376, entitled“Mast Cell Growth Factor” published on Jan. 9, 1992 and assigned to theImmunex Corporation or alternatively in European Patent Application No423 980, entitled “Stem Cell Factor” published April 24, 1992 andassigned to Amgen Inc) was present in the suspension culture phase onlya minor amplification of progenitor cell production occurred (FIG. 26)but when combined with GM-CSF, IL-3, or IL-1, 200- to 800-foldamplification occurred. The combination of IL-1, KL and either GM-CSF orIL-3 was even more effective in amplifying progenitors, and the fourfactor combination of IL-1+KL+IL-6 with either IL-3 or GM-CSF producedup to 2,500-fold increases in progenitor cells. Calculations ofprogenitor cell generation based on CFU-GM output.HPP-CFC input showedthat three factor combinations (IL-1+KL_IL-3 or CSF's) generated ratiosof 6,000 to 10,000 and four factor combinations (including IL-6)generated ratios of 8,000 to 15,000. As measure of self-renewal thegeneration of secondary HPP-CFC-1 as a ratio of HPP-CFC input reachedvalues of 50 to 700 with two factor combinations of KL with IL-1, IL-3or CFS's and 700 to 1,300 with three factor combinations of IL-1+KL withIL-6, IL-3, or CSFs.

[0268] Based on the total differentiating cells produced in a 7-dayculture of enriched HPP-CFC exposed to a combination of IL-1 plus IL-3plus KL, FIG. 27 illustrates the dramatic proliferation obtained. Thisincludes a self-renewal component measured by secondary HPP-CFC-1generation, a progenitor cell production measured by low proliferativepotential CFU-GM, and morphologically identifiable differentiatingmyeloid cells. The cell population doubling time required to generatethese cells from a single precursor reaches the limits of knownmammalian cell proliferation rates. If this proliferation was sustainedby an earlier even more infrequent cell than the HPP-CFC, an evenshorter population doubling time would be required. The amplification ofHPP-CFC in this short-term culture is unlikely to be reflected in acomparable expansion in long-term reconstituting cells, and the majorityof HPP-CFC, an even shorter population doubling time would be required.

[0269] The amplification of HPP-CFC is unlikely to be reflected in acomparable expansion in long-term reconstituting cells, and the majorityof HPP-CFC generated are more likely to representative of later stageswithin the stem cell hierarchy. Assay of D12 CFU-S also showed anabsolute increase in numbers after 7 days suspension culture with IL-1plus IL-3 or KL. Other investigators have shown that in similarsuspension cultures, precursors of CFU-GEMM (possibly long-termreconstituting stem cells) also amplified in the presence of IL-1 plusIL-3 but not with IL-6 and IL-3 or GM-CSF combinations.

[0270] Delta or secondary CFU assay for early hematopoietic cells: Humanstudies. In humans, 4-HC treatment of bone marrow has been shown todeplete the majority of progenitors capable of responding directly toGM-CSF by in vitro colony formation while preserving stem cells capableof colony formation while preserving stem cells capable of hematopoieticreconstitution in the context of bone marrow transplantation. Inprimitive transplantation studies, CD34⁺ selection also enriched formarrow cells capable of long-term reconstitution. Following combine 4-HCtreatment and selection of CD34⁺ cells by immunocytoadherence, primarycolony formation in response to G-CSF or GM-CSF was extremely low.However, 7 days of suspension culture followed by secondary recloningwith FM-CSF showed that exposure of treated marrow cells for 7 days insuspension to combination of IL-1 and IL-3 consistently generated thehighest numbers of secondary CFU-GM. IL-3 and IL-6 was no less effectivethan IL-3 alone and other cytokine combinations were significantly lesseffective. Secondary colony formation in this assay was maximallystimulated by combinations of IL-1 and KL, KL and IL-3, and combinationsof all three cytokines was most effective in amplifying progenitor cellgeneration.

[0271] Interactions Between c-kit Ligand (KL) and IL-18, IL-6 and otherHematopoietic Factors

[0272] The in vivo purging of BM with 5-FU is a simple technique for theenrichment of quiescent hematopoietic progenitor cells. A single dose of5-FU can, within 24 hours, reduce the numbers of early-appearing CFU-Sand the more mature CFU-C populations by greater than 99%, whileenriching the BM for more primitive progenitors. Late-appearing CFU-Sare also sensitive to BM purging with 5-FU, further suggesting thatthese cells are not he same as stem cell responsible for long-term BMreconstitution. In contrast, BM reconstituting stem cells have beenshown to be refractory to the cytotoxic effects of 5-FU(105). Bradleyand Hodgson, using 5-FU purged BM, identified a compartment ofprogenitor cells, HPP-CFC, that are capable of forming large highlycellular colonies in agar cultures.

[0273] We have investigated the interactions of IL-1, IL-6 and KL onprimitive murine progenitor cell compartments (104). We presentevidence, using clonal cultures, for synergistic and additive effects ofthese factors alone or in conjunction with CSF's. Our results suggestthat IL-1, IL-6 and KL act uniquely in their stimulation of earlyhematopoiesis. The finding with the clonal cultures are furthersubstantiated using a short-term liquid culture assay, the Δ-assay, thathas been previously described. We demonstrate the ability of IL-1, IL-6and KL and regulate the expansion of early and late hematopoieticprogenitor compartments.

Materials and Methods

[0274] Mice. Male and female (C57BL/6×DBA/2)F₁ (B6D2F1) mice werepurchased from The Jackson Laboratory (Bar Harbor, Me.). The mice weremaintained under laminar-flow conditions, and were provided withacidified and/or autoclaved drinking water. Sentinel mice, housed alongwith the colony, were observed for specific pathogens. All mice usedwere of at least 8 weeks of age.

[0275] Marrow Preparation and Tissue Culture Conditions. BM from normal(NBM) or 5-FU treated mice was obtained from femora and sometimes tibiaof at least 3 mice per experiment. Mice were treated with 5-FU byintravenous injection of 150 mg/kg in a volume of 150 to 250 μl. BM waswashed twice by centrifugation before culturing. Unless otherwise noted,all handling and cultures of BM was done in culture medium containingIMDM (Gibco, Grand Island, N.Y.) supplemented with 20% FCS (HyCloneLaboratories Inc., Logan Utah) and 0.05% mg/ml gentamicin (Gibco). BMcells were enumerated using a Coulter counter model ZBI (coulterElectronics, Hialeah, Fla.). All plasticware used was of tissue culturegrade.

[0276] Cytokines and Antibodies. Purified rhIL-11, sp act=1.32×10₇U/mg,(Syntex Laboratories, Inc.,: Palo Alto, Calif.) was used at 100 U/ml.Partially purified and purified rhIL-6 was kindly provided by StevenGillis (Imunex Corporation, Seattle, Wash.); partially purified IL-6 wasused at 3000 CESS U/ml and purified IL-6 was used at 50 ng/ml. PurifiedKL (prepared as described herein or alternatively prepared as describedin PCT International Publication No. WO 92/00376, entitled “Mast CellGrowth Factor” published on Jan. 9, 1992 and assigned to the ImmunexCorporation or alternatively in European Patent Application No 423 980,entitled “Stem Cell Factor” published Apr. 24, 1992 and assigned toAmgen Inc). Purified rhG-CSF (Amgen Biologicals, Thousand Oaks, Calif.)was used at 1000 U/ml (sp act=1×10 108 U/mg). Purified rhM-CSF was usedat 1000 U/ml (Immunex). Conditioned media containing rmIL-3 was preparedfrom transiently transfected COS-7 cells, and like all other growthfactors was used at concentrations resulting in maximal CFU-Cstimulation. Rat anti-mouse IL-6 monoclonal antibody was purchased fromGenzyme (Cambridge, Mass.).

[0277] CFU-C Assay. LPP-CFC was assayed in 35 mm petri dishes containing1 ml of 5×10⁴ NBM suspended in culture medium containing cytokines and0.36% agarose (SeaPlaque; FMC, Rockland, Me.). Such cultures wereincubated for 7 days at 37° C. in a fully humidified 5% C02 atmosphere.HPP-CFC were assayed using a double-layer agarose system previouslydescribed. Sixty mm petri dishes containing a 2 ml underlayer consistingof culture media, cytokines and 0.5% agarose was overlayed with 1 ml of5-FU 1 to 8 days prior (d1 -d8 5-FU BM) was assayed for HPP-CFC at cellconcentrations ranging from 1×10³ to 1×10⁵ cells/culture. Double-layercultures were grown for 12 days at 37° C. in a fully humidified, 5% CO2,and 7% O2 atmosphere. Dishes were scored for low proliferative coloniescontaining at least 50 cells (LPP-CFC) and highly cellular highproliferative colonies with diameters of at least 0.5 mm (HPP-CFC). AllCFU-C were enumerated from triplicate cultures.

[0278] CFU-S Assay. Mice were irradiated with 1250 Gy from a 137Cs γ-raysource at a dose rate of approximately 90 Gy/minute. The 1250 Gy wasgiven as a split dose of 800 Gy plus 450 Gy separated by 3 hours. BMcells were injected intravenously 2-3 hours after the final irradiation.Late-appearing CFU-S were counted on spleens fixed in Bouin's solution12 days after BM transplantation.

[0279] Delta (Δ) Assay. Suspension cultures were performed as previouslydescribed. Quadruplicate 1 ml Δ-cultures consisting of 2.5×10₅ d1 5-FUBM cells/ml were established in 24 well cluster plates and incubated inthe presence of growth factors for 7 days at 37° C. in fully humidified5% CO2 atmosphere/Non-adherent cells from week old cultures wereharvested after vigorous pipetting. Resuspended BM cells fromquadruplicate Δ-cultures were pooled and 1 ml was used for thedetermination of culture cellularity. The remaining 3 ml of cells werewashed by centrifugation through and underlayer of 5 ml FCS. Washedcells were assayed for secondary LPP-CFC, HPP-CFC and CFU-S. SecondaryLPP-CFC responsive to G-CSF, GM-CSF and IL-3 were measured in 7 dayCFU-C cultures. Secondary HPP-CFC and LPP-CFC responsive to IL-1 andIL-3 were enumerated after 12 days under the conditions described forgrowth of HPP-CFC. Cells from Δ-cultures were diluted from 20 to2,000-fold for the determination of secondary CFU-C. The numbers ofCFU-S present in Δ-cultures after one week's growth were determined bytransplanting mice with 2 to 200-fold dilutions of washed cells.

[0280] The fold increases in BM progenitor populations after Δ-culturehas been termed the Δ-value. The numbers of primary LPP-CFC, HPP-CFC andCFU-S present in the starting d1 5-FU BM population were measured inparallel to the suspension cultures. Delta-values were determined bydividing the total output of secondary LPP-CFC, HPP-CFC and CFU-S by theinput of primary LPP-CFC, HPP-CFC and CFU-S respectively.

[0281] Adherent-cell Depleted Δ-Assay. Delta-cultures, of 12.5 ml of2.5×10⁵ d1 5-FU BM cells/ml, were established in 25 cm² tissue cultureflasks. Before the onset of culture, BM was depleted of adherent cellpopulations by a single 4 hour incubation at 37° C. in culture medium.Non-adherent cells were transferred to a second 25 cm₂ flask, and bothcell populations were maintained under the conditions described abovefor Δ-cultures.

[0282] Assays for Cytokine Activity. Delta-culture supernatants, fromcultures grown in 25 cm² tissue culture flasks, were collected bycentrifugation. Supernatants were collected from cultures establishedwith dl 5-FU BM, adherent cell depleted BM and BM adherent cells. IL-6activity was measured using the murine hybridoma B9 cell proliferationassay as previously described. Cytokine activity was also measured usingthe growth dependent hematopoietic cell line NFS-60. Proliferation ofNFS-60 cells in response to growth factor activity was measured aspreviously described.

[0283] Statistics. Significance was determined using the two-way pairedStudent's t-test.

Results

[0284] Activities of IL-1, IL-6, and KL on NBM. The effects of G-CSF,M-CSF, GM-CSF and IL-3 in combination of IL-1, IL-6 and KL on colonyformation from NBM is shown in FIG. 1. Colony formation in response toIL-1, IL-6, KL and IL-1 plus IL-6 was minimal. Combining the stimulus ofIL-1 with M-CSF, GM-CSF or IL-4 increased colony formation over thatobserved with the CSF's alone, most notably the greater than additiveeffects of IL-1 and M-CSF stimulation which was consistently seen inrepeated studies. The addition of IL-6 to CSF-containing culturesincreased colony formation in an additive fashion. The combined stimulusof IL-1 plus IL-6, alone or in combination with the CSF's, did notnoticeably affect colony growth in a greater than additive fashion. Theaddition of KL to IL-1, IL-6, G-CSF, GM-CSF or IL-3 containing culturesstimulated CFU-C in a synergistic manner. KL did not synergize withM-CSF. The addition of CSF-to IL-1 plus KL or IL-6 plus KL-stimulatedcultures demonstrated additive or less than additive colony growth.

[0285] Activities of IL-1, IL-6 and KL on 5-FU BM. The recovery ofHPP-CFC and LPP-CFC from 1 to 7 days after a single administration of5-FU to mice is shown in FIGS. 2 and 3. Few colonies grew in response toIL-1 and/or IL-6 stimulation, although several HPP-CFC as well asLPP-CFC were consistently detected. The lineage restricted CSF's, G-CSFand M-CSF, had little ability to stimulate HPP-CFC, whereas GM-CSF andIL-3 were able to stimulate both HPP-CFC and LPP-CFC. The greateststimulation of-HPP-CFC required combinations of growth factors.

[0286] Kit-Ligand had almost no detectable colony-stimulating activity,with only an average of 1.3 HPP-CFC and 2.7 LPP-CFC being stimulatedfrom 1×10⁴d7 5-FU BM cells (FIG. 30). The concentration of KL usedthroughout most of this study was 20 ng/ml. This concentration of KL topromote high proliferative colony formation in the presence of IL-1 andIL-6. At 1 ng/ml KL an average of 6.7 colonies were observed, whereasfrom 10 to 100 ng/ml KL colony numbers reached a plateau in the range of120 to 147 HPP-CFC per 2.5×10⁴ d4 5-FU BM cells (data not shown). Theaddition of KL to G-CSF containing cultures resulted in increasednumbers of HPP-CFC in d1 5-FU BM as well as increase number of LPP-CFCin both d1 and d7 5-FU BM populations. Synergism among KL and G-CSF instimulating HPP-CFC was pronounce in cultures of d4 5-FU BM (data notshown). The combination of KL plus M-CSF did not result in anysuper-additive colony formation. However KL showed strong synergism instimulating HPP-CFC in the presence of GM-CSF and IL-3. IL-3 plus KL wasa more effective stimulus of large colony formation that IL-1 plus IL-3in both d1 and d7 5-FU BM populations; addition of KL to IL-3 containingcultures increased the numbers of HPP-CFC by 6 to 35 fold in d1 and d75-FU BM respectively.

[0287] Although IL-1, IL-6 or KL have no appreciable CSF activity, theaddition of KL to IL-1, IL-6 or IL-1 plus IL-6 containing culturesresults in dramatize synergism among these factors in promoting thegrowth of HPP-CFC (FIG. 30). Combining KL with IL-6 or IL-1 stimulatedan average of 4.0 and 13.7 high proliferative colonies of 1×10⁵ d1 5-FUBM cells respectively. Moreover, in response to all three cytokines anaverage of 42.0 HPP-CFC per 1×10⁵ cells were stimulated. These resultsclearly demonstrate the existence of a subpopulation of HPP-CFC thatrequire stimulation of IL-1, IL-6 plus KL for large colony formation.The response of d7 5-FU B<to these growth factor combinations wassimilar to dl 5-FU BM to these growth factor combinations was similar todi 5-FU B</However, the proportion of HPP-CFC stimulated with IL-1, IL-6plus KL in d7 5-FU BM was less than a tenth of the maximum number ofHPP-CFC that could be stimulated by the further addition of GM-CSF tothis three factor combination. The difference in the dl 5-FU BMpopulation was less dramatic with the maximum number of HPP-CFCstimulated by four cytokines being only a little more than twice thenumber stimulated by IL-1, IL-6 plus KL. The addition of IL-6 tocultures containing combinations of KL and CSF's did not enhance largecolony formation above the numbers that could be accounted for by theadditive effects of two factor combinations of IL-6, KL and CSF (FIG.30). For instance, the combination of IL-6, KL plus GM-CSF resulted inapproximately 30 high proliferative colonies per 1×10⁵ d1 5-FU BM cells.The bulk of these 30 HPP-CFC could be accounted for by the combinednumber of colonies observed in IL-6 plus KL plus GM-CSF-stimulatedcultures (4 and 20 HPP-CFC respectively), suggesting that IL-6, KL plusCSF do not combine to recruit any additional HPP-CFC to proliferative.

[0288] In contrast to the above results with IL-6, the addition of IL-1to cultures containing KL and CSF did demonstrate synergism (FIG. 30).This synergism was most evident in the cultures of d7 5-FU BM grown incombinations of IL-1, KL plus G-CSF. Any two factor combination of thesethree cytokines stimulated 5 or less HPP-CFC, whereas the combination ofIL-1, KL plus G-CSF resulted in an average of 100 HPP-CFC per 1×10⁴ BMcells. Although not as pronounced, synergism was evident among IL-1, KLplus GM-CSF or IL-1 in stimulating d7 5-FU BM. These super-additiveeffects were also apparent in the dl 5-FU BM population withcombinations of IL-1, KL plus G-CSF or M-CSF. The large number ofHPP-CFC present in dl 5-FU BM stimulated by combinations of IL-1, KLplus GM-CSF or IL-3 could, however, be attributed to additive effects ofthese growth factors on different populations of HPP-CFC.

[0289] As mentioned above, the greatest number of HPP-CFC werestimulated by combinations of four growth facts, with the stimuli IL-1,IL-6, KL plus GM-CSF or IL-3 being optimal (FIG. 30). The combination ofIL-1 IL-6, KL plus GM-CSF was capable of stimulating over 3% of d7 5-FUBM cells to form high proliferative colonies. Only with the cytokinemixture of IL-1 IL-6, KL plus M-CSF did the observed increase in HPP-CFCappear to be due to synergism of all four growth factors in promotingadditional large colony growth not observed with combinations of fewercytokines. The addition of IL-6 to the cytokine combinations of IL-1, KLplus G-CSF, GM-CSF or IL-3 did not result in superadditive colonyformation. The number of high proliferative colonies stimulated by IL-1,IL-6, KL plus G-CSF, GM-CSF, or IL-3 were, in most cases, notsignificantly greater than the number of HPP-CFC stimulated with thecombinations IL-1, KL plus G-CSF, GM-CSF, or IL-3.

[0290] Expansion of 5-FU BM in Δ-Cultures. The numbers of non-adherentcells recovered after 7 days of growth in Δ-cultures reflected thepattern of response observed with various combinations of cytokines inthe clonal cultures of 5-FU BM (FIG. 31). Control cultures of d15-FU BMreceiving no cytokine stimulation had an average 39% decline in culturecellularity, with the predominant surviving cell population beingmonocyte/macrophage. The addition of IL-1, IL-6 or KL alone did notincrease the recovery of cells above the input level. Except for slightincreases in response to GM-CSF and IL-3, only those cultures stimulatedwith multiple cytokines expanded their cell numbers. The greatestproliferation resulted from cultures stimulated with IL-1, KL plusGM-CSF or IL-3, the further addition of IL-6 to these cultures did notincrease the recovery of cells significantly. The appearance of immaturemyeloid cells correlated with the observed proliferation of theΔ-cultures. In one experiment, IL-3 stimulated cultures contained about50% mature segmented neutrophils and macrophages, 25% metamyelocytes,20% myelocyte and 3% blast cells. The percentage of blast cells increasewith the addition of IL-1)22%), IL-6(18%), KL(24t), IL-1 plus IL-6(12%),IL-1 plus KL(51%), IL-6 plus KL(42%) and IL-1, IL-6 plus KL(46%) to IL-3containing cultures. The greatest total number of blast cells, 6.1×105cells, was recovered from cultures stimulated with IL-1, KL and IL-3,representing on the order of a 200 fold increase over the startingd15-FU BM population.

[0291] Control Δ-cultures, grown without the addition of cyrokines, didnot increase LPP-CFC progenitor cell populations over input values (FIG.5). Expansion was evident with the addition of the colony-stimulatingfactors G-CSF, M-CSF, GM-CSF and IL-3 (mean Δ-values of 3.4, 2.4, 23 and140 respectively). IL-1 alone stimulated over a sixty-fold increase inLPP-CFC, and combining the stimuli of IL-1 and CSF's resulted insynergistic expansions of LPP-CFC. For example, IL-1 plus IL-3 had amean Δ-value of 520 as compared to the predicted additive Δ-value of140(TL-3)+63(IL-1)=203. IL-6 stimulated a small but significantexpansion of LPP-CFC (Δ-value=3.4; p<0.01). Greater than additiveeffects were evident in the combination of IL-6 plus G-CSF and IL-6 plusIL-3. KL did not significantly increase the recovery of LPP-CFC fromΔ-cultures (p-0.08). The combined stimuli of KL and CSF's was, however,greater than additive in all cases. The combination KL plus IL-3 was aseffective as IL-1 plus IL-3 in expanding LPP-CFC (mean Δ-value=485 and520 respectively; p=0.21). Delta-cultures stimulated with IL-1 plus IL-6in combination with CSF's had higher Δ-values in all cases than culturesstimulated with IL-1 or IL-6. The increased LPP-CFC expansion wasadditive in all combinations of IL-1, IL-6 plus CSF except in culturesstimulated with IL-1, IL-6 plus M-CSF (Δ-value=300, compared to IL-1plus M-CSF, Δ-value=140, or IL-6 plus M-CSF, Δ-value=2.8). IL-6 plus KLwas synergistic in stimulating the expansion of LPP-CFC over 200-fold,however the addition of these two cytokines to CSF containing culturesresulted in only additive increases in progenitor cells. Together, IL-1and KL were synergistic in stimulating over a 1,000-fold expansion inLPP-CFC. The addition of G-CSF, GM-CSF or IL-3 to IL-1 plusKL-containing cultures further increased the expansion of LPP-CFC (meanΔ-values of 1100, 1200 and 1400 respectively). The greatest expansion ofLPP-CFC was achieved with combinations of IL-1, IL-6, KL plus CSF's.Delta-cultures stimulated with IL-1, IL-6, KL plus IL-3 had over an1,800-fold expansion of LPP-CFC. Although increasing the Δ-values, theaddition of IL-6 to IL-1 plus KL-containing Δ-cultures did notsignificantly add to the observed progenitor cell expansion (p>0.05).

[0292] Expansion of HPP-CFC in Δ-Cultures. The ability of differentcytokine combinations to stimulate the expansion of HPP-CFC was tested(FIG. 33). As was the case with the expansion of LPP-CFC, the greatestincreases in HPP-CFC evident in Δ-cultures stimulated with combinationsof IL-1, KL plus CSF. Alone, the CSF's stimulated only a modest increasein HPP-CFC. IL-6 stimulated an increase in HPP-CFC, furthermore thecombined stimulation of IL-6 plus IL-3 was more effecting in expandingMPP-CFC than IL-3 alone. In contrast to IL-6, IL-1 demonstratedsynergism in combination with all four CSF's. KL, in combination withall four CSF's, also stimulated the expansion of HPP-CFC in a greaterthan additive fashion. The combination of IL-1 plus IL-6, with orwithout CSF's, was more effective in expanding HPP-CFC than either IL-1or IL-6 alone. The clearest case of synergism using IL-1 plus IL-6 wasin combination with M-CSF (mean Δ-values of 1.0 with IL-6+M-CSF, 13.2with IL-1+M-CSF and 65.7 with IL-1+IL-6-M-CSF). The addition of IL-1 orIL-6 to Δ-cultures containing KL, alone or in combination with CSF'sresulted in greater than additive increases in HPP-CFC. Althoughincreasing the Δ-values in each case, the addition of CSF's to culturescontaining KL with either IL-2 or IL-6 did not significantly increasethe expansion of HPP-CFC. The greatest expansion of HPP-CFC was incultures stimulated with IL-1, IL-6 plus KL (Δ-value of 705).

[0293] Secondary HPP-CFC produced in Δ-cultures are routinely assayed inclonal assays stimulated with IL-1 plus IL-3 (FIG. 33). Othercombination of cytokines, such as IL-1 plus GM-CSF or IL-1 plus M-CSF,have been tested for their ability to stimulate secondary HPP-CFC. Theenumeration of secondary HPP-CFC grown in the presence of IL-1 plusM-CSF or GM-CSF was hindered due to the abundance of secondary LPP-CFC,relative to the number of HPP-CFC, stimulated by these cytokinecombinations. The effectiveness of IL-1 and KL as a stimulus forsecondary HPP-CFC was also tested (FIG. 34). In contrast to any othercombination of cytokines tested, IL-1 plus KL-responsive progenitorcells did not expand dramatically in Δ-cultures that did stimulate theexpansion of IL-1 plus IL-3-responsive HPP-CFC and LPP-CFC.

[0294] Expansion of CFU-S in Δ-Cultures. In an effort to furthercharacterize the populations of BM cells that emerge after Δ-cultures,we examined the increase in CFU-S in response to cytokine stimulation inΔ-cultures (FIG. 35). Cultures grown in the presence of IL-1, IL-3, IL-1plus IL-3 or IL-1 plus KL demonstrated increases in HPP-CFC and LPP-CFCconsistent with the results presented in FIGS. 32 and 33. These culturesalso exhibited increases in CFU-S that were greater than the increasesin HPP-CFC. IL-1 plus IL-3 and IL-1 plus KL stimulated over 100-foldexpansion in the number of late-appearing CFU-S. These results werecompared to the expansion of HPP-CFC and CFU-S that are known to occurin mice recovering from 5-FU treatment; the in vivo expansion (Δ invivo) was measured by dividing the total femoral HPP-CFC, LPP-CFC andCFU-S in d8 5-FU BM by the total numbers of colonies observed per d15-FU femur. The in vivo expansion of progenitor cells was similar tothat observed in in vitro Δ-cultures, with the exception that theincrease in LPP-CFC in vivo was less than those observed in vitro.

Discussion

[0295] These studies substantiate the roles of IL-1, IL-6 and KL asregulators of primitive hematopoietic cells. Alone, these cytokines havea limited ability to stimulate the proliferation of murine hematopoieticprogenitor cells in our clonal culture assays (FIGS. 29-30). However,synergism among IL-1, IL-6 and KL was evident in the stimulation ofcolony growth. By systematic analysis in combinations of IL-1, IL-6, KLplus colony-stimulating factors we were able to discriminate populationsof HPP-CFC and LPP-CFC present in 5-FU purged BM. The ability of IL-1,IL-6 and/or KL to regulate colony formation by primitive hematopoieticcells was also supported by experiments employing short-term liquidcultures of dl 5-FU BM. The Δ-assay, which is capable of measuring theflux in progenitor populations in response to cytokine stimulation,demonstrated that the greatest expansion of LPP-CFC and HPP-CFC wasdependent upon the synergistic interactions of IL-1, IL-6, KL and CSF'son early hematopoietic progenitors (FIGS. 32-35).

[0296] The importance of IL-1 as a regulator of early hematopoiesis hasbeen known since its identification as the synergistic activity,Hemopoietin-1, present in the conditioned medium of the bladdercarcinoma cell line 5637. Consistent with previously reported results,we have shown IL-1 to synergize with G-CSF, M-CSF, GM-CSF, IL-1 or KL inthe stimulation of HPP-CFC (FIGS. 29 and 30). The ability of IL-1 topromote the proliferation of primitive hematopoietic cells was alsoobserved in the Δ-assay (FIGS. 31-33). The synergistic activity of IL-1,in combination with G-CSF, M-CSF, GM-CSF, IL-3 or KL, was manifest inits ability to promote the expansion of the total number of cells, thenumber of myeloid blast cells, the number of LPP-CFC and the number ofHPP-CFC in liquid culture. Several studies have suggested that thecytokine combination IL-1 plus IL-3 G-CSF, M-CSF, GM-CSF. In Δ-cultures,the stimulus IL-1 plus IL-3 was capable of expanding LPP-CFC and HPP-CFCby 520 and 83-fold respectively, this expansion of progenitorpopulations was greater than those stimulated by IL-1 plus G-CSF, M-CSFor GM-CSF. However, the synergism observed between IL-1 and KL was amore effective stimulus than IL-1 plus IL-3 in the expansion of dl 5-FUBM.

[0297] Delta-cultures stimulated with IL-1 plus KL increased the numberof LPP-CFC by over 1000-fold and the number of HPP-CFC by 280 fold.

[0298] The hematopoietic activities of IL-6 were found to differ fromthose of IL-1. The combinations IL-6 plus IL-3 or KL were found to besynergistic in the stimulation of HPP-CFC from d1 -d7 5-FU BM (FIGS.30). IL-6 and KL were also synergistic in the stimulation of CFU-C fromNBM (FIG. 28). In the Δ-assay, synergism was evident between IL-6 andeither IL-3 or KL in the expansion of LPP-CFC and HPP-CFC (FIGS. 5 and6). IL-6 plus IL-3 was not as effective as IL-1 plus IL-3 in theexpansion of HPP-CFC (Δ-values=40 and 83 respectively). The three factorcombination of IL-1, IL-6 and M-CSF was found to be synergistic instimulating HPP-CFC from d1 -d7 5-FU BM. Furthermore, the Δ-assay alsodemonstrated synergism in the expansion of LPP-CFC and HPP-CFCpopulations in response to IL-1, IL-6 plus M-CSF. The cytokinecombination of IL-1, IL-6 plus KL was synergistic in stimulating thegrowth of HPP-CFC from d1 and d7 5-FU BM. The addition of IL-1, IL-6plus KL to Δ-cultures also resulted in the greatest observed expansionof HPP-CFC (Δ-value=705). These patterns of synergistic interactionsamong IL-1, IL-6, KL and CSF's demonstrate the unique roles of IL-1,IL-6 and KL in the regulation of pluripotential hematopoieticprogenitors.

[0299] The stimulatory effects of KL upon early hematopoieticprogenitors observed in this study are in accord with the stem cellgrowth activity that was instrumental in the cloning of the KL gene. Theresponse of NBM progenitors to IL-1, IL-6, -G-CSF, GM-CSF or IL-1demonstrated synergism in combination with KL (FIG. 28). As previouslyreported, KL did not enhance colony formation in response to M-CSF fromNBM. The same pattern of response was observed using 5-FU BM; KL wassynergistic with IL-1, IL-6, G-CSF, GM-CSF or IL-3, but not with M-CSF(FIG. 30). The dramatic synergism in the stimulation of HPP-CFC observedwith IL-1 plus KL could be further augmented by the addition of CSF's.Most notable was the synergism observed among IL-1, KL and G-CSF incultures of d1 and d7 5-FU BM. The optimal hematopoietic response wasobserved with the four cytokine combinations of IL-1, IL-6, KL plus CSF.only with the combination IL-1, IL-6, KL plus M-CSF was the four growthfactor stimulation of HPP-CFC synergistic. The combinations IL-1, IL-6,KL plus GM-CSF or IL-3 stimulated the most HPP-CFC, the greatestproliferation of cells in Δ-cultures and the largest expansion ofLPP-CFC in Δ-cultures (FIGS. 31-33). These results demonstrate theimportance of KL in the regulation of the proliferation of earlyhematopoietic cells.

[0300] HPP-CFC represent a hierarchy of cells that can be distinguishedbased on their growth factor requirements and/or physical separationtechniques. The identification of two compartments of earlyhematopoietic cells, HPP-CFC-1 and HPP-CFC-2, correlates with theseparation of progenitor cells based on their retention of themitochondrial dye rhodamine-123. Rhodamine-123 dull cells represent themore primitive HPP-CFC-1 compartment of cells that require thesynergistic interactions of IL-1, IL-3 and M-CSF for theirproliferation, whereas the HPP-CFC-2 compartment of cells do not requirestimulation by IL-1. The more primitive nature of IL-1 plus CSFstimulated progenitor cells is in agreement with the synergisticinteraction observed with IL-1 and CSF's in the expansion of LPP-CFC andHPP-CFC in the Δ-assay (FIGS. 32 and 33). Furthermore, the regulation ofprimitive hematopoietic cells is also governed by the growth factorsIL-6 and KL. The ability of IL-6 and KL to expand HPP-CFC in Δ-culturesis suggestive of their role in the stimulation of progenitor cells thatare considered to be HPP-CFC-1. These data support the contention thatquiescent stem cells, that are spared by 5-FU purging of BM, requirestimulation by multiple growth factors for their proliferation. Thematuration of these progenitor cells, from Hpp-CFC-1 to HPP-CFC-1, isfollowed by a restriction in the requirement for multiple-cytokinestimulated proliferation. Consistent with the concept of a hierarchy ofHPP-CFC is the observation that over 3% of d7 5-FU BM cells are capableof forming HPP-CFC in response to IL-1, IL-6, KL plus GM-CSF stimulation(FIG. 30), an incidence far higher than the estimate frequency oftotipotential stem cells present in the BM.

[0301] The increase of HPP-CFC in Δ-cultures is suggestive of anexpansion of multipotential hematopoietic progenitors. However, theplacement of these post Δ-culture HPP-CFC in the hierarchy of HPP-CFC isunclear. The observed increases in late-appearing CFU-S in Δ-culturessupports the contention that the number of multipotential hematopoieticprogenitors are expanded under the conditions of the Δ-assay (FIG. 35).CFU-S were increased over 100-fold in response to Il-1 plus IL-3 or KLplus IL-l or IL-3 stimulated suspension cultures of purifiedrhodamine-123 bright or dull progenitor cells. Our results are contraryto the reported decline in CFU-S in liquid cultures of d2 5-FU BMstimulated wit IL-6 plus IL-3 or KL may be more advantageous in gentherapy protocols. Our Results also suggest that the expansion ofprogenitor cells with the cytokines IL-1 plus IL-3 or KL may bebeneficial in bone marrow transplantation protocols. HPP-CFC responsiveto IL-1 plus KL were minimally expanded by combinations of the growthfactors IL-1, IL-3, IL-6 and KL in Δ-cultures (FIG. 34). The ability ofIL-1 plus KL to promote the growth of HPP-CFC from 5-FU BM as well asstimulate large increases in progenitor cells in the Δ-assay isindicative of the ability of IL-1 plus KL to act upon a pool orprimitive multipotential progenitors. The limited expansion of IL-1 plusKL responsive HPP-CFC is suggestive of a limited ability of the growthfactors IL-1, IL-3, IL-6 and KL to stimulate the self-renewal of earlyhematopoietic progenitors and stem cells in the Δ-assay.

[0302] IL-1 and KL Induced Proliferation and the Influence of TGFB andMIP1α.

[0303] TGFB and MIP1α Macrophage Inflammatory Protein-1α have beenpreviously reported to inhibit progenitors. Such reports have suggestedthat either of these cytokines might act as a negative regulator ofhematopoietic stem call proliferation, although the two have notpreviously been compared directly in recognized stem cell assays. Themurine HPP colony assay assesses stem cell properties by depleting laterprogenitors with 5-fluorouracil and scoring only colonies with highproliferative potential as assessed by size (>0.5=m). IL-1and KLpreferentially stimulate early hematopoietic progenitors. We thereforechose to evaluate the effects of TGFB and MIP1α on HPP proliferationinduced by IL-1 and KL/Results from two separate experiments, eachperformed in triplicate, are expressed as HPP colony numbers induced bythe growth factor combinations shown relative to those induced by GM-CSF(GM) alone: IL−1 + GM IL−1 + GM KL + GM KL + GM IL−1 + KL Control 1.0 ±.1 7.0 ± 1.3 3.9 ± .8 47.3 ± 6.5  9.7 ± 1.5 TCFB1 1.2 ± .2 1.3 ± 0.5 1.4± .2 2.0 ± 0.2 0 ± 0 TGFB3 1.0 ± .2 1.3 ± 0.1 1.1 ± .3 1.4 ± 0.2 0 ± 0MIP1α 0.9 ± .2 6.9 ± 0.7 6.3 ± .6 50.8 ± 6.5  15.8 ± 2.1 

[0304] These results demonstrate that TGFB abrogates the synergisticproliferation of HPP colonies promoted by IL-1 and/or KL with GM-CSF,whereas MIP1α has no such effect. Furthermore TGFB eliminated HPPcolonies induced by IL-1+k1α, whereas MIP1α actually promoted HPP colonyformation under these conditions. We conclude that TGFB, but not MIP1,acts as a negative regulator of the hematopoietic progenitor populationsassessed here. This has important implications for the design ofchemotherapy protection protocols.

[0305] Studies of KL in Combination with IL-3, EPO or GM-CSF

[0306] 11 patients with DBA, al prednisone resistant or requiring highdoses, had decreased mean BFU-E frequency with rhEpo and rhIL-stimulation. With the exception of one prednisone sensitive patient,these values were below the 95% confidence limit obtained from 4 normaladult bone marrows. When recombinant murine cKit ligand (rmKL) waseither added to or substituted for rhIL- all patients showed significantincrease in BFU-E size and hemoglobinization. Moreover, the combinationof rhEPO, rhIL- and rmKL at least double mean BFU-E frequency in 8 or 11patients (range: 2 to 16 fold). RhIL3-induced myeloid colonies were alsodecreased to <95% confidence limit in 5 of the 11 patients. The additionof KL increased man myeloid colony frequency 2 fold or greater in 6patients.

[0307] BFU-E stimulated with rhEpo plus rhIL-3 and/or rmKL wereundetectable in 6 FA patients with various degrees of bone marrowinsufficiency. Myeloid colonies were also undetectable in 4 cases, andsignificantly decreased in 2 with either rhIL-3 or rhGM-CSF stimulation.The addition of rmKL or rhIL-3 increased mean frequency in the latter.RhIL3 plus rmKL induced myeloid colonies in a third patient with DC, onewith more sever aplasia had no erythroid or myeloid colonies with eitherrhIL-3 or rhGM-CSF alone or with rmKL, the second patient had adecreased mean BFU-E frequency with rhEpo and rhIL-3 (13% of normalcontrol). BFU-E from the latter patient increased in size,hemoglobinization and number with the addition of rmKL. RhIL-3 orrhGM-CSF-stimulated myeloid colonies were slightly decreased and KLinduced an appropriate increase in mean colony frequency.

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1 15 1 825 DNA Artificial Sequence KL cDNA Clone 1 gcggtgcctt tccttatgaagaagacacaa acttggatta tcacttgcat ttatcttcaa 60 ctgctcctat ttaatcctctcgtcaaaacc aaggagatct gcgggaatcc tgtgactgat 120 aatgtaaaag acattacaaaactggtggca aatcttccaa atgactatat gataaccctc 180 aactatgtcg ccgggatggatgttttgcct agtcattgtt ggctacgaca tatggtaata 240 caattatcac tcagcttgactactcttctg gacaagttct caaatatttc tgaaggcttg 300 agtaattact ccatcatagacaaacttggg aaaatagtgg atgacctcgt gttatgcatg 360 gaagaaaacg caccgaagaatataaaagaa tctccgaaga ggccagaaac tagatccttt 420 actcctgaag aattctttagtattttcaat agatccattg atgcctttaa ggactttatg 480 gtggcatctg acactagtgactgtgtgctg tcttcaacat taggtcccga gaaagattcc 540 agagtcagtg tcacaaaaccatttatgtta ccccctgttg cagccagctc ccttaggaat 600 gacagcagta gcagtgataggaaagccgca aagtcccctg aagactcggg cctacaatgg 660 acagccatgg cattgccggctctcatttcg cttgtaattg gctttgcttt tggagcctta 720 tactggaaga agaaacagtcaagtcttaca agggcagttg aaaatataca gattaatgaa 780 gaggataatg agataagtatgctgcaacag aaagagagag aattt 825 2 270 PRT Artificial Sequence PredictedSequence of KL cDNA clone 2 Met Lys Lys Thr Gln Thr Trp Ile Ile Thr CysIle Tyr Leu Gln Leu 1 5 10 15 Leu Leu Phe Asn Pro Leu Val Lys Thr LysGlu Ile Cys Gly Asn Pro 20 25 30 Val Thr Gln Met Val Lys Gln Ile Thr LysLeu Val Ala Asn Leu Pro 35 40 45 Asn Asp Tyr Asn Ile Thr Leu Met Tyr ValAla Gly Asn Asp Val Leu 50 55 60 Pro Ser Asn Cys Trp Leu Arg Asp Asn ValIle Gln Leu Ser Leu Ser 65 70 75 80 Leu Thr Thr Leu Leu Asp Lys Phe SerAsn Ile Ser Glu Gly Leu Ser 85 90 95 Met Tyr Ser Ile Ile Asp Lys Leu GlyLys Ile Val Asp Gln Leu Val 100 105 110 Leu Cys Met Glu Glu Asn Ala ProLys Asn Ile Lys Glu Ser Pro Lys 115 120 125 Arg Pro Glu Thr Arg Ser PheThr Pro Glu Glu Phe Phe Ser Ile Phe 130 135 140 Asn Arg Ser Ile Asp AlaPhe Lys Asp Phe Met Val Ser Ser Asp Thr 145 150 155 160 Ser Asp Cys ValLeu Ser Ser Thr Leu Gly Pro Glu Lys Asp Ser Arg 165 170 175 Val Ser ValThr Lys Pro Phe Met Leu Pro Pro Val Ala Ala Ser Ser 180 185 190 Leu ArgAsn Asp Ser Ser Ser Ser Asn Arg Lys Ala Ala Lys Ser Pro 195 200 205 GluAsp Ser Gly Leu Gln Trp Thr Ala Asn Ala Leu Pro Ala Leu Ile 210 215 220Ser Leu Val Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp Lys Lys Lys 225 230235 240 Gln Ser Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu245 250 255 Cys Asn Glu Ile Ser Met Leu Gln Gln Lys Glu Arg Glu Phe 260265 270 3 1344 DNA Artificial Sequence KL cDNA 3 gggactatct gcagccgctgctggtgcaat atgctggagc tccagaacag ctaaacggag 60 tcgccacacc gctgcctgggctggatcgca gcgctgcctt tccttatgaa gaagacacaa 120 acttggatta tcacttgcatttatcttcaa ctgctcctat ttaatcctct tgtcaaaacc 180 aaggagatct gcgggaatcctgtgactgat aatgtaaaag acattacaaa actggtggca 240 aatcttccaa atgactatatgataaccctc aactatgtcg ccgggatgga tgttttgcct 300 agtcattgtt ggctacgagatatggtaata caattatcac tcagcttgac tactcttctg 360 gacaagttct caaatatttctgaaggcttg agtaattact ccatcataga caaacttggg 420 aaaatagtgg atgacctcgtgttatgcatg gaagaaaacg caccgaagaa tataaaagaa 480 tctccgaaga ggccagaaactagatccttt actcctgaag aattctttag tattttcaat 540 agatccattg atgcctttaaggactttatg gtggcatctg acactagtga ctgtgtgctc 600 tcttcaacat taggtcccgagaaagattcc agagtcagtg tcacaaaacc atttatgtta 660 ccccctgttg cagccagctcccttaggaat gacagcagta gcagtaatag gaaagccgca 720 aaggcccctg aagactcgggcctacaattg acagccatgg cattgccggc tctcatttcg 780 cttgtaattg gctttgcttttggagcctta tactggaaga agaaacagtc aagtcttaca 840 agggcagttg aaaatatacagattaatgaa gaggataatg agataagtat gttgcaacag 900 aaagagagag aatttcaagaggtgtaattg tggacgtatc aacattgtta ccttcgcaca 960 gtggctggta acagttcatgtttgcttcat aaatgaagca gccttaaaca aattcccatt 1020 ctgtctcaag tgacagacctcatccttacc tgttcttgct acccgtgacc ttgtgtggat 1080 gattcagttg ttggagcagagtgcttcgct gtgaaccctg cactgaatta tcatctgtaa 1140 agaaaaatct gcacggagcaggactctgga ggttttgcaa gtgatgatag ggacaagaac 1200 atgtgtccag tctacttgcaccgtttgcat ggcttgggaa acgtctgagt gctgaaaacc 1260 cacccagctt tgttcttcagtcacaacctg cagcctgtcg ttaattatgg tctctgcaag 1320 tagatttcag cctggatggtgggg 1344 4 273 PRT Artificial Sequence Predicted Sequence of KL cDNA 4Met Lys Lys Thr Gln Thr Trp Ile Ile Thr Cys Ile Tyr Leu Gln Leu 1 5 1015 Leu Leu Phe Asn Pro Leu Val Lys Thr Lys Glu Ile Cys Gly Asn Pro 20 2530 Val Thr Asp Asn Val Lys Asp Ile Thr Lys Leu Val Ala Asn Leu Pro 35 4045 Asn Asp Tyr Met Ile Thr Leu Asn Tyr Val Ala Gly Met Asp Val Leu 50 5560 Pro Ser His Cys Trp Leu Arg Asp Met Val Ile Gln Leu Ser Leu Ser 65 7075 80 Leu Thr Thr Leu Leu Asp Lys Phe Ser Asn Ile Ser Glu Gly Leu Ser 8590 95 Asn Tyr Ser Ile Ile Asp Lys Leu Gly Lys Ile Val Asp Asp Leu Val100 105 110 Leu Cys Met Glu Glu Asn Ala Pro Lys Asn Ile Lys Glu Ser ProLys 115 120 125 Arg Pro Glu Thr Arg Ser Phe Thr Pro Glu Glu Phe Phe SerIle Phe 130 135 140 Asn Arg Ser Ile Asp Ala Phe Lys Asp Phe Met Val AlaSer Asp Thr 145 150 155 160 Ser Asp Cys Val Leu Ser Ser Thr Leu Gly ProGlu Lys Asp Ser Arg 165 170 175 Val Ser Val Thr Lys Pro Phe Met Leu ProPro Val Ala Ala Ser Ser 180 185 190 Leu Arg Asn Asp Ser Ser Ser Ser AsnArg Lys Ala Ala Lys Ala Pro 195 200 205 Glu Asp Ser Gly Leu Gln Trp ThrAla Met Ala Leu Pro Ala Leu Ile 210 215 220 Ser Leu Val Ile Gly Phe AlaPhe Gly Ala Leu Tyr Trp Lys Lys Lys 225 230 235 240 Gln Ser Ser Leu ThrArg Ala Val Glu Asn Ile Gln Ile Asn Glu Glu 245 250 255 Asp Asn Glu IleSer Met Leu Gln Gln Lys Glu Arg Glu Phe Gln Glu 260 265 270 Val 5 27 DNAArtificial Sequence Primer 5 gcccaagctt cggtgccttt ccttatg 27 6 36 DNAArtificial Sequence Primer 6 agtatctcta gaattttaca cctcttgaaa ttctct 367 33 DNA Artificial Sequence Primer 7 catttatcta gaaaacatga actgttaccagcc 33 8 24 DNA Artificial Sequence Primer 8 accctcgagg ctgaaatcta cttg24 9 40 PRT Murinae gen. sp. MISC_FEATURE (4)..(4) X = UNKNOWN RESIDUE 9Lys Glu Ile Xaa Gly Asn Pro Val Thr Asp Asn Val Lys Asp Ile Thr 1 5 1015 Lys Leu Val Ala Asn Leu Pro Asn Asp Tyr Met Ile Thr Leu Asn Tyr 20 2530 Val Ala Gly Met Xaa Val Leu Pro 35 40 10 93 DNA Artificial SequencecDNA 10 aagcttgata acgtraaaga tatcacaaaa ctggtggcaa atcttccaaatgactatatg 60 ataaccctca attacgtggc gggcatggga tcc 93 11 30 DNAArtificial Sequence Sense Primer 11 cgccaagctt gayaaygtna argayathac 3012 28 DNA Artificial Sequence Antisense Primer 12 ttratrcanc gnccntaccctaggggcc 28 13 67 DNA Artificial Sequence S1d cDNA insert 13 gtctctctttgacaaggtgg agaagtcact gatgactgga gaaaggcttg gctctatcat 60 tgacaga 67 1493 DNA Artificial Sequence cDNA 14 aagcttgata atgtaaaaga cattacaaaactggtggcaa atcttccaaa tgactatatg 60 ataaccctca attacgtggc cggaatggga tcc93 15 93 DNA Artificial Sequence cDNA 15 aagcttgata atgttaaagacataacaaaa ctggtggcaa atcttccaaa tgactatatg 60 ataaccctca actacgtagccggcatggga tcc 93

What is claimed is:
 1. A pharmaceutical composition which comprises aneffective amount of c-kit ligand and an effective amount of ahematopoietic factor or factors in a suitable pharmaceutical carrier. 2.A pharmaceutical composition for enhancing engraphment of bone marrowduring transplantation in a mammal which comprises the composition ofclaim 1 wherein the hematopoietic factor is IL-1, in an amount effectiveto enhance engraftment of bone marrow during transplantation in amammal.
 3. A pharmaceutical composition for enhancing bone marrowrecovery in treatment of radiation, chemical or chemotherapeutic inducedbone marrow aplasia or myelosuppression which comprises the compositionof claim 1 wherein the hematopoietic factor is IL-1, in an amounteffective to enhance bone marrow recovery in a mammal.
 4. Apharmaceutical composition for the treatment of acute myelogenousleukemia in a mammal which comprises the composition of claim 1 whereinthe hematopoietic factor is GM-CSF, in an amount effective to treatacute myelogenous leukemia in a mammal.
 5. A pharmaceutical compositionfor the treatment of chronic myelogenous leukemia in a mammal whichcomprises the composition of claim 1 wherein the hematopoietic factor isGM-CSF, in an amount effective to treat chronic myelogenous leukemia ina mammal.
 6. A method for treating leukemia in a patient which comprisesadministering to the patient the pharmaceutical composition of claim 1wherein the hematopoietic factor is GM-CSF, in an amount effective toincrease white blood cells vulnerability to chemotherapy and therebytreat leukemia in the patient.
 7. A pharmaceutical composition forstimulation of progenitor cells in a patient which comprises thecomposition of claim 1 in an amount effective to stimulate theprogenitor cells.
 8. The pharmaceutical composition of claim 7, whereinthe hematopoietic factor is IL-1.
 9. The pharmaceutical composition ofclaim 7, wherein the hematopoietic factor is IL-3.
 10. Thepharmaceutical composition of claim 7, wherein the hematopoietic factoris IL-6.
 11. The pharmaceutical composition of claim 7, wherein thehematopoietic factors are IL-1 and IL-6.
 12. The pharmaceuticalcomposition of claim 7, wherein the hematopoietic factors are IL-1 andIL-3.
 13. The pharmaceutical composition of claim 7, wherein thehematopoietic factors are IL-1 and GM-CSF.
 14. The pharmaceuticalcomposition of claim 7, wherein the hematopoietic factors are IL-1 andMIP1α.
 15. The pharmaceutical composition of claim 7, wherein thehematopoietic factors are IL-1, IL-6 and IL-3.
 16. The pharmaceuticalcomposition of claim 7, wherein the hematopoietic factors are IL-!, IL-6and GM-CSF.
 17. A pharmaceutical composition for increasing levels ofstem cells in peripheral blood which comprises the composition of claim1 wherein the hematopoietic factor is IL-1, in an amount effective tocause stem cells to enter the peripheral blood.
 18. A method forincreasing levels of stem cells in peripheral blood which comprisesadministering to a mammal the pharmaceutical composition of claim 17 toincrease the levels of stem cells in peripheral blood.
 19. Apharmaceutical composition of claim for treatment of leucopenia in amammal which comprises the composition of claim 1 wherein thehematopoietic factor is selected from the group consisting of G-CSF,GM-CSF and IL-3, in an amount effective to treat leucopenia in a mammal.20. An antagonist of c-kit ligand which comprises a soluble, mutatedc-kit ligand which is capable of binding to a c-kit receptor but doesnot cause biological activity which occurs when normal, functioningc-kit ligand binds to the c-kit receptor.
 21. An antagonist of c-kitligand which comprises a small molecule which is capable of binding to ac-kit receptor but does not cause biological activity which occurs whennormal, functioning c-kit ligand binds to the c-kit receptor.
 22. Anantisense nucleic acid molecule capable of binding to c-kit ligand mRNAand preventing translation of the c-kit ligand mRNA.
 23. Apharmaceutical composition for treating leukemia in a mammal whichcomprises an effective amount of the pharmaceutical composition of claim20 to treat leukemia.
 24. A method of treating melanoma in a patientwhich comprises administering to the patient an effective amount of thecomposition of claim 20 to treat melanoma.
 25. A pharmaceuticalcomposition for the treatment of allergies in a patient which comprisesan effective amount of the pharmaceutical-composition of claim 20 inaerosol form to treat allergies.
 26. A pharmaceutical composition forthe treatment of asthma in a patient which comprises an effective amountof the pharmaceutical composition of claim 20 in aerosol form to treatasthma.
 27. A pharmaceutical composition for the treatment of rheumatoidarthritis in a patient which comprises an effective amount of thepharmaceutical composition of claim 20 in topical form to treatrheumatoid arthritis.
 28. A pharmaceutical composition for the treatmentof a dermal allergic reaction in a patient which comprises an effectiveamount of the pharmaceutical composition of claim 20 in topical form totreat the dermal allergic reaction.
 29. The pharmaceutical compositionof claim 28, wherein the dermal allergic reaction is scleroderma.
 30. Apharmaceutical composition for the treatment of allergic conjunctivitisin a patient which comprises an effective amount of the pharmaceuticalcomposition of claim 20 to treat allergic conjunctivitis.
 31. Apharmaceutical composition for protection against anaphylaxic shock in apatient which comprises an effective amount of the pharmaceuticalcomposition of claim 20 to protect the patient from anaphylaxic shock.32. A pharmaceutical composition for blocking a histamine mediatedresponse which comprises an effective amount of the composition of claim20 to inhibit mast cell production and thereby block the histaminemediated response.
 33. The pharmaceutical composition of claim 32,wherein the histamine mediated response is secretion of gastric acid byparietal cells.
 34. A pharmaceutical composition for blockingpost-allergic tissue damage which comprises an effective amount of thecomposition of claim 20 to inhibit mast cell production and therebyreduce mast cell secretion of proteases and subsequent post-allergictissue damage.
 35. A composition which comprises c-kit ligand and anappropriate carrier suitable for ex-vivo use.
 36. A method for enhancingtransfection of early hematopoietic progenitor cells with a gene whichcomprises: a) contacting early hematopoietic cells with the compositionof claim 35 and a hematopoietic factor forming cultured cells; b) andtransfecting the cultured cells of step (a) with the gene.
 37. Themethod of claim 36, wherein the gene encodes for antisense RNA.
 38. Amethod of transferring a gene to a mammal which comprises: a) contactingearly hematopoietic progenitor cells with the composition of claim 35;b) transfecting the calls of (a) with the gene; and c) administering thetransfected cells of (b) to the mammal.
 39. The method of claim 38,wherein the gene encodes for antisense RNA.
 40. A composition forexpansion of peripheral blood levels ex-vivo which comprises thecomposition of claim 35 and an effective amount of a hematopoieticgrowth factor or factors, in an amount effective to expand theperipheral blood levels ex-vivo.
 41. A pharmaceutical composition ofclaim 40, wherein the hematopoietic growth factor is IL-1.
 42. Thepharmaceutical composition of claim 40, wherein the hematopoietic factoris IL-3.
 43. The pharmaceutical composition of claim 40, wherein thehematopoietic factor is IL-6.
 44. The pharmaceutical composition ofclaim 40, wherein the hematopoietic factors are IL-1 and IL-6.
 45. Thepharmaceutical composition of claim 40, wherein the hematopoieticfactors are IL-1 and IL-3.
 46. The pharmaceutical composition of claim40, wherein the hematopoietic factors are IL-1 and GM-CSF.
 47. Thepharmaceutical composition of claim 40, wherein the hematopoieticfactors are IL-1 and MIP1α.
 48. The pharmaceutical composition of claim40, wherein the hematopoietic factors are IL-1, IL-6 and IL-3.
 49. Thepharmaceutical composition of claim 40, wherein the hematopoieticfactors are IL-1, IL-6 and GM-CSF.
 50. A method for ex-vivo expansion ofperipheral blood levels which comprises treating cells ex-vivo with thecomposition of claim 35 and an effective amount of a hematopoieticgrowth factor, effective to expand the peripheral blood levels ex-vivo.51. A method for increasing platelet levels in peripheral blood levelwhich comprises treating cells with the composition of claim 35 incombination with another hematopoietic factor, effective to boost theplatelet levels in peripheral blood ex-vivo.
 52. A method of claim 50,wherein the hematopoietic factor is Il-6.
 53. A method of modifying abiological function associated with c-kit cellular activity whichcomprises contacting a cell, whose function is to be modified, with thecomposition of claim 35, effective to modify the biological function ofthe cell.
 54. The method of claim 53, wherein the biological function isthe propagation of a cell that expresses c-kit.
 55. The method of claim53, wherein the cell which expresses c-kit is a hematopoietic cell. 56.The method of claim 53, wherein the biological function is in vitrofertilization.
 57. A method of modifying a biological functionassociated with c-kit cellular activity in a patient which comprisesadministering to the patient an effective amount of c-kit ligandeffective to modify the biological function associated with c-kitfunction.
 58. A method according to claim 57, wherein the biologicalfunction is inducing differentiation of erythroid progenitors.
 59. Amethod according to claim 57, wherein the biological function istreating infants exhibiting symptoms of defective lung development. 60.A method according to claim 57, wherein the biological function isincreasing the pigmentation in the person's hair.
 61. A method accordingto claim 57, wherein the biological function is improving neuronsurvival.
 62. A pharmaceutical composition which comprises an effectiveamount of c-kit ligand in a suitable pharmaceutical carrier.
 63. Amethod for the treatment of anemia in a patient which comprisesadministering in a patient an effective amount of the composition ofclaim 62 to treat the anemia.
 64. A method for enhancing engraftment ofbone marrow during transplantation in a patient which comprisesadministering an effective amount of the composition of claim 62 toenhance engraphment of bone marrow.
 65. A method of enhancing bonemarrow recovery in treatment of radiation, chemical, or chemotherapeuticinduced bone marrow aplasia or myelosuppression which comprises treatingpatients with therapeutic effective doses of the composition of claim 62to enhance the bone marrow recovery.
 66. A method for the treatingacquired immune deficiency in a patient which comprises administering tothe patient a therapeutically effective amount of the composition ofclaim 62 to treat the acquired immune deficiency.
 67. A pharmaceuticalcomposition for inducing differentiation of erythroid progenitors in apatient which comprises an effective amount of the composition of claim62 to induce differentiation of the erythroid progenitors.
 68. Acomposition for treating infants exhibiting symptoms of defective lungdevelopment which comprises an effective amount of the composition ofclaim 62 to treat infants exhibiting symptoms of defective lungdevelopment.
 69. A composition for increasing pigmentation in asubject's hair, which comprises an effective amount of the compositionof claim 62 to increase the pigmentation in the subjects hair.
 70. Amethod for the treatment of leucopenia in a patient which comprisesadministering an effective amount the composition of claim 62 to treatthe leukopenia.